CA2551462A1 - Methods for nucleic acid isolation and kits using solid phase material - Google Patents
Methods for nucleic acid isolation and kits using solid phase material Download PDFInfo
- Publication number
- CA2551462A1 CA2551462A1 CA002551462A CA2551462A CA2551462A1 CA 2551462 A1 CA2551462 A1 CA 2551462A1 CA 002551462 A CA002551462 A CA 002551462A CA 2551462 A CA2551462 A CA 2551462A CA 2551462 A1 CA2551462 A1 CA 2551462A1
- Authority
- CA
- Canada
- Prior art keywords
- nucleic acid
- sample
- solid phase
- phase material
- inhibitors
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 407
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 407
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 407
- 238000000034 method Methods 0.000 title claims abstract description 327
- 239000000463 material Substances 0.000 title claims description 617
- 239000007790 solid phase Substances 0.000 title claims description 343
- 238000002955 isolation Methods 0.000 title description 32
- 239000000523 sample Substances 0.000 claims abstract description 465
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 217
- 239000012472 biological sample Substances 0.000 claims abstract description 12
- 239000003112 inhibitor Substances 0.000 claims description 246
- 230000002934 lysing effect Effects 0.000 claims description 119
- 238000000576 coating method Methods 0.000 claims description 110
- 239000011248 coating agent Substances 0.000 claims description 107
- 210000004027 cell Anatomy 0.000 claims description 105
- 230000008569 process Effects 0.000 claims description 89
- 239000004094 surface-active agent Substances 0.000 claims description 89
- -1 polytetrafluoroethylene Polymers 0.000 claims description 78
- 239000002245 particle Substances 0.000 claims description 73
- 239000011159 matrix material Substances 0.000 claims description 62
- 238000011068 loading method Methods 0.000 claims description 50
- 239000002736 nonionic surfactant Substances 0.000 claims description 48
- 238000002156 mixing Methods 0.000 claims description 47
- 229920000867 polyelectrolyte Polymers 0.000 claims description 44
- 238000000926 separation method Methods 0.000 claims description 44
- 230000003321 amplification Effects 0.000 claims description 37
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 37
- 230000004888 barrier function Effects 0.000 claims description 32
- 229920001343 polytetrafluoroethylene Polymers 0.000 claims description 32
- 239000004810 polytetrafluoroethylene Substances 0.000 claims description 32
- 210000000170 cell membrane Anatomy 0.000 claims description 28
- 238000006243 chemical reaction Methods 0.000 claims description 26
- 238000010438 heat treatment Methods 0.000 claims description 11
- 239000002699 waste material Substances 0.000 claims description 5
- 239000000243 solution Substances 0.000 description 154
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 111
- 108020004414 DNA Proteins 0.000 description 85
- 210000004369 blood Anatomy 0.000 description 84
- 239000008280 blood Substances 0.000 description 84
- 210000004940 nucleus Anatomy 0.000 description 78
- 239000012528 membrane Substances 0.000 description 75
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 60
- 238000000605 extraction Methods 0.000 description 59
- 239000002585 base Substances 0.000 description 47
- 239000011324 bead Substances 0.000 description 43
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 41
- 210000000265 leukocyte Anatomy 0.000 description 33
- 239000000203 mixture Substances 0.000 description 26
- 241000700605 Viruses Species 0.000 description 23
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 22
- 108090000623 proteins and genes Proteins 0.000 description 21
- 238000003260 vortexing Methods 0.000 description 21
- 210000003743 erythrocyte Anatomy 0.000 description 20
- 239000000872 buffer Substances 0.000 description 19
- 241000894006 Bacteria Species 0.000 description 17
- 239000006228 supernatant Substances 0.000 description 17
- 230000009089 cytolysis Effects 0.000 description 16
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 15
- 238000010828 elution Methods 0.000 description 15
- 102000004169 proteins and genes Human genes 0.000 description 15
- 230000001580 bacterial effect Effects 0.000 description 14
- 125000000524 functional group Chemical group 0.000 description 14
- 238000012545 processing Methods 0.000 description 14
- 239000011521 glass Substances 0.000 description 13
- 229920000642 polymer Polymers 0.000 description 13
- 238000002360 preparation method Methods 0.000 description 13
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 12
- 150000003278 haem Chemical class 0.000 description 12
- 239000012071 phase Substances 0.000 description 12
- 230000003612 virological effect Effects 0.000 description 12
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 11
- 150000003839 salts Chemical class 0.000 description 11
- 239000013612 plasmid Substances 0.000 description 10
- 238000005119 centrifugation Methods 0.000 description 9
- 210000002966 serum Anatomy 0.000 description 9
- 238000005406 washing Methods 0.000 description 9
- WMFOQBRAJBCJND-UHFFFAOYSA-M Lithium hydroxide Chemical compound [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 description 8
- 125000000217 alkyl group Chemical group 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 238000010790 dilution Methods 0.000 description 8
- 239000012895 dilution Substances 0.000 description 8
- 239000000835 fiber Substances 0.000 description 8
- 239000012530 fluid Substances 0.000 description 8
- 239000007787 solid Substances 0.000 description 8
- 238000009987 spinning Methods 0.000 description 8
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 7
- 229920002125 Sokalan® Polymers 0.000 description 7
- 239000000377 silicon dioxide Substances 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 239000002888 zwitterionic surfactant Substances 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 239000007983 Tris buffer Substances 0.000 description 6
- MCMNRKCIXSYSNV-UHFFFAOYSA-N Zirconium dioxide Chemical compound O=[Zr]=O MCMNRKCIXSYSNV-UHFFFAOYSA-N 0.000 description 6
- 239000012670 alkaline solution Substances 0.000 description 6
- 239000012141 concentrate Substances 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 6
- 210000002381 plasma Anatomy 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical group N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 5
- 229910019142 PO4 Inorganic materials 0.000 description 5
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 5
- 150000001298 alcohols Chemical class 0.000 description 5
- 239000003945 anionic surfactant Substances 0.000 description 5
- 210000001124 body fluid Anatomy 0.000 description 5
- 239000010839 body fluid Substances 0.000 description 5
- 230000001413 cellular effect Effects 0.000 description 5
- 239000013039 cover film Substances 0.000 description 5
- 239000007857 degradation product Substances 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000006260 foam Substances 0.000 description 5
- NBZBKCUXIYYUSX-UHFFFAOYSA-N iminodiacetic acid Chemical group OC(=O)CNCC(O)=O NBZBKCUXIYYUSX-UHFFFAOYSA-N 0.000 description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000006166 lysate Substances 0.000 description 5
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 5
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 5
- 235000021317 phosphate Nutrition 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 230000002441 reversible effect Effects 0.000 description 5
- 150000003512 tertiary amines Chemical class 0.000 description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 5
- 108091005804 Peptidases Proteins 0.000 description 4
- 102000035195 Peptidases Human genes 0.000 description 4
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 4
- 239000003093 cationic surfactant Substances 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- 239000012149 elution buffer Substances 0.000 description 4
- 230000002209 hydrophobic effect Effects 0.000 description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 4
- 229910052742 iron Inorganic materials 0.000 description 4
- 229910052751 metal Inorganic materials 0.000 description 4
- 239000002184 metal Substances 0.000 description 4
- 150000003141 primary amines Chemical class 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 235000019833 protease Nutrition 0.000 description 4
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 4
- 150000003335 secondary amines Chemical class 0.000 description 4
- 125000001424 substituent group Chemical group 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 229920002554 vinyl polymer Polymers 0.000 description 4
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 108010067770 Endopeptidase K Proteins 0.000 description 3
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical group C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 102000001554 Hemoglobins Human genes 0.000 description 3
- 108010054147 Hemoglobins Proteins 0.000 description 3
- 101001102158 Homo sapiens Phosphatidylserine synthase 1 Proteins 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical group C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 102100039298 Phosphatidylserine synthase 1 Human genes 0.000 description 3
- 239000004698 Polyethylene Substances 0.000 description 3
- 239000004793 Polystyrene Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 239000007984 Tris EDTA buffer Substances 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 150000003973 alkyl amines Chemical group 0.000 description 3
- 150000008051 alkyl sulfates Chemical class 0.000 description 3
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 3
- 150000001412 amines Chemical class 0.000 description 3
- 235000019270 ammonium chloride Nutrition 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-O ammonium group Chemical group [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000005349 anion exchange Methods 0.000 description 3
- 125000000129 anionic group Chemical group 0.000 description 3
- 150000001450 anions Chemical class 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 125000004432 carbon atom Chemical group C* 0.000 description 3
- 125000002091 cationic group Chemical group 0.000 description 3
- 150000001768 cations Chemical class 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000006037 cell lysis Effects 0.000 description 3
- 229920001429 chelating resin Polymers 0.000 description 3
- 238000004891 communication Methods 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 230000002779 inactivation Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 230000002458 infectious effect Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 159000000003 magnesium salts Chemical class 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical group OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 description 3
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 239000005022 packaging material Substances 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 229920000768 polyamine Polymers 0.000 description 3
- 229920000573 polyethylene Polymers 0.000 description 3
- 229920000098 polyolefin Polymers 0.000 description 3
- 229920002223 polystyrene Polymers 0.000 description 3
- 125000001453 quaternary ammonium group Chemical group 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000005204 segregation Methods 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 229920001169 thermoplastic Polymers 0.000 description 3
- MBYLVOKEDDQJDY-UHFFFAOYSA-N tris(2-aminoethyl)amine Chemical group NCCN(CCN)CCN MBYLVOKEDDQJDY-UHFFFAOYSA-N 0.000 description 3
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 2
- CHRJZRDFSQHIFI-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;styrene Chemical compound C=CC1=CC=CC=C1.C=CC1=CC=CC=C1C=C CHRJZRDFSQHIFI-UHFFFAOYSA-N 0.000 description 2
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- DVLFYONBTKHTER-UHFFFAOYSA-N 3-(N-morpholino)propanesulfonic acid Chemical compound OS(=O)(=O)CCCN1CCOCC1 DVLFYONBTKHTER-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- 239000005695 Ammonium acetate Substances 0.000 description 2
- 101001007348 Arachis hypogaea Galactose-binding lectin Proteins 0.000 description 2
- 102000004506 Blood Proteins Human genes 0.000 description 2
- 108010017384 Blood Proteins Proteins 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 206010058279 Factor V Leiden mutation Diseases 0.000 description 2
- 229910017974 NH40H Inorganic materials 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- ABLZXFCXXLZCGV-UHFFFAOYSA-N Phosphorous acid Chemical compound OP(O)=O ABLZXFCXXLZCGV-UHFFFAOYSA-N 0.000 description 2
- 239000004743 Polypropylene Substances 0.000 description 2
- GOOHAUXETOMSMM-UHFFFAOYSA-N Propylene oxide Chemical group CC1CO1 GOOHAUXETOMSMM-UHFFFAOYSA-N 0.000 description 2
- 241000191940 Staphylococcus Species 0.000 description 2
- 241000194017 Streptococcus Species 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 241000726445 Viroids Species 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- MGSKVZWGBWPBTF-UHFFFAOYSA-N aebsf Chemical compound NCCC1=CC=C(S(F)(=O)=O)C=C1 MGSKVZWGBWPBTF-UHFFFAOYSA-N 0.000 description 2
- 235000019257 ammonium acetate Nutrition 0.000 description 2
- 229940043376 ammonium acetate Drugs 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000003575 carbonaceous material Substances 0.000 description 2
- 238000005341 cation exchange Methods 0.000 description 2
- 230000003196 chaotropic effect Effects 0.000 description 2
- 230000009920 chelation Effects 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 239000000084 colloidal system Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- 238000003851 corona treatment Methods 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 229920002313 fluoropolymer Polymers 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 108010036302 hemoglobin AS Proteins 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 229910010272 inorganic material Inorganic materials 0.000 description 2
- 239000011147 inorganic material Substances 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 239000003456 ion exchange resin Substances 0.000 description 2
- 229920003303 ion-exchange polymer Polymers 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- IBIKHMZPHNKTHM-RDTXWAMCSA-N merck compound 25 Chemical compound C1C[C@@H](C(O)=O)[C@H](O)CN1C(C1=C(F)C=CC=C11)=NN1C(=O)C1=C(Cl)C=CC=C1C1CC1 IBIKHMZPHNKTHM-RDTXWAMCSA-N 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 125000000962 organic group Chemical group 0.000 description 2
- 239000011368 organic material Substances 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 238000005191 phase separation Methods 0.000 description 2
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 2
- 229920001983 poloxamer Polymers 0.000 description 2
- 229920000515 polycarbonate Polymers 0.000 description 2
- 239000004417 polycarbonate Substances 0.000 description 2
- 229920001155 polypropylene Polymers 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 239000003161 ribonuclease inhibitor Substances 0.000 description 2
- 210000003296 saliva Anatomy 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000002594 sorbent Substances 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 239000012536 storage buffer Substances 0.000 description 2
- 150000003871 sulfonates Chemical class 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- JNYAEWCLZODPBN-JGWLITMVSA-N (2r,3r,4s)-2-[(1r)-1,2-dihydroxyethyl]oxolane-3,4-diol Chemical compound OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O JNYAEWCLZODPBN-JGWLITMVSA-N 0.000 description 1
- LPJKDVHMUUZHRY-KVVVOXFISA-N (z)-octadec-9-en-1-amine;hydrochloride Chemical compound Cl.CCCCCCCC\C=C/CCCCCCCCN LPJKDVHMUUZHRY-KVVVOXFISA-N 0.000 description 1
- QLAJNZSPVITUCQ-UHFFFAOYSA-N 1,3,2-dioxathietane 2,2-dioxide Chemical compound O=S1(=O)OCO1 QLAJNZSPVITUCQ-UHFFFAOYSA-N 0.000 description 1
- FJLUATLTXUNBOT-UHFFFAOYSA-N 1-Hexadecylamine Chemical compound CCCCCCCCCCCCCCCCN FJLUATLTXUNBOT-UHFFFAOYSA-N 0.000 description 1
- FKKAGFLIPSSCHT-UHFFFAOYSA-N 1-dodecoxydodecane;sulfuric acid Chemical class OS(O)(=O)=O.CCCCCCCCCCCCOCCCCCCCCCCCC FKKAGFLIPSSCHT-UHFFFAOYSA-N 0.000 description 1
- NLMKTBGFQGKQEV-UHFFFAOYSA-N 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-hexadecoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical compound CCCCCCCCCCCCCCCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO NLMKTBGFQGKQEV-UHFFFAOYSA-N 0.000 description 1
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 description 1
- BMUDPLZKKRQECS-UHFFFAOYSA-K 3-[18-(2-carboxyethyl)-8,13-bis(ethenyl)-3,7,12,17-tetramethylporphyrin-21,24-diid-2-yl]propanoic acid iron(3+) hydroxide Chemical compound [OH-].[Fe+3].[N-]1C2=C(C)C(CCC(O)=O)=C1C=C([N-]1)C(CCC(O)=O)=C(C)C1=CC(C(C)=C1C=C)=NC1=CC(C(C)=C1C=C)=NC1=C2 BMUDPLZKKRQECS-UHFFFAOYSA-K 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical class OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- 101000766308 Bos taurus Serotransferrin Proteins 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- QYOVMAREBTZLBT-KTKRTIGZSA-N CCCCCCCC\C=C/CCCCCCCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO Chemical compound CCCCCCCC\C=C/CCCCCCCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO QYOVMAREBTZLBT-KTKRTIGZSA-N 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 108010038061 Chymotrypsinogen Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- QRLVDLBMBULFAL-UHFFFAOYSA-N Digitonin Natural products CC1CCC2(OC1)OC3C(O)C4C5CCC6CC(OC7OC(CO)C(OC8OC(CO)C(O)C(OC9OCC(O)C(O)C9OC%10OC(CO)C(O)C(OC%11OC(CO)C(O)C(O)C%11O)C%10O)C8O)C(O)C7O)C(O)CC6(C)C5CCC4(C)C3C2C QRLVDLBMBULFAL-UHFFFAOYSA-N 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical class NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 208000037357 HIV infectious disease Diseases 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical group Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 229920001732 Lignosulfonate Polymers 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229920001410 Microfiber Polymers 0.000 description 1
- 108091092878 Microsatellite Proteins 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- UEEJHVSXFDXPFK-UHFFFAOYSA-N N-dimethylaminoethanol Chemical compound CN(C)CCO UEEJHVSXFDXPFK-UHFFFAOYSA-N 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 102000007999 Nuclear Proteins Human genes 0.000 description 1
- 108010089610 Nuclear Proteins Proteins 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000012807 PCR reagent Substances 0.000 description 1
- 108091093037 Peptide nucleic acid Proteins 0.000 description 1
- 229920002845 Poly(methacrylic acid) Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229920002873 Polyethylenimine Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 229930003427 Vitamin E Natural products 0.000 description 1
- AWSYOWHJNGZJGU-OASARBKBSA-N [(2r,3s,4s,5s)-3,4-dihydroxy-5-(hydroxymethyl)-5-[(2r,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxolan-2-yl]methyl octanoate Chemical compound O[C@H]1[C@H](O)[C@@H](COC(=O)CCCCCCC)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 AWSYOWHJNGZJGU-OASARBKBSA-N 0.000 description 1
- ZAKOWWREFLAJOT-ADUHFSDSSA-N [2,5,7,8-tetramethyl-2-[(4R,8R)-4,8,12-trimethyltridecyl]-3,4-dihydrochromen-6-yl] acetate Chemical group CC(=O)OC1=C(C)C(C)=C2OC(CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C ZAKOWWREFLAJOT-ADUHFSDSSA-N 0.000 description 1
- JNGWKQJZIUZUPR-UHFFFAOYSA-N [3-(dodecanoylamino)propyl](hydroxy)dimethylammonium Chemical compound CCCCCCCCCCCC(=O)NCCC[N+](C)(C)[O-] JNGWKQJZIUZUPR-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000000159 acid neutralizing agent Substances 0.000 description 1
- 150000003926 acrylamides Chemical class 0.000 description 1
- 150000001252 acrylic acid derivatives Chemical class 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000000274 adsorptive effect Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 229910001413 alkali metal ion Inorganic materials 0.000 description 1
- 150000004996 alkyl benzenes Chemical class 0.000 description 1
- 229940045714 alkyl sulfonate alkylating agent Drugs 0.000 description 1
- 150000008052 alkyl sulfonates Chemical class 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- BTBJBAZGXNKLQC-UHFFFAOYSA-N ammonium lauryl sulfate Chemical compound [NH4+].CCCCCCCCCCCCOS([O-])(=O)=O BTBJBAZGXNKLQC-UHFFFAOYSA-N 0.000 description 1
- 229940063953 ammonium lauryl sulfate Drugs 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 229940019748 antifibrinolytic proteinase inhibitors Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 239000004760 aramid Substances 0.000 description 1
- 229920006231 aramid fiber Polymers 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- YOUGRGFIHBUKRS-UHFFFAOYSA-N benzyl(trimethyl)azanium Chemical group C[N+](C)(C)CC1=CC=CC=C1 YOUGRGFIHBUKRS-UHFFFAOYSA-N 0.000 description 1
- 229960003237 betaine Drugs 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000003613 bile acid Substances 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 150000005323 carbonate salts Chemical class 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 150000001733 carboxylic acid esters Chemical class 0.000 description 1
- 239000000679 carrageenan Substances 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 229940113118 carrageenan Drugs 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 210000002230 centromere Anatomy 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 229960002887 deanol Drugs 0.000 description 1
- DTPCFIHYWYONMD-UHFFFAOYSA-N decaethylene glycol Polymers OCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO DTPCFIHYWYONMD-UHFFFAOYSA-N 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- UVYVLBIGDKGWPX-KUAJCENISA-N digitonin Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)C[C@@H](O)[C@H](O[C@H]5[C@@H]([C@@H](O)[C@@H](O[C@H]6[C@@H]([C@@H](O[C@H]7[C@@H]([C@@H](O)[C@H](O)CO7)O)[C@H](O)[C@@H](CO)O6)O[C@H]6[C@@H]([C@@H](O[C@H]7[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O7)O)[C@@H](O)[C@@H](CO)O6)O)[C@@H](CO)O5)O)C[C@@H]4CC[C@H]3[C@@H]2[C@@H]1O)C)[C@@H]1C)[C@]11CC[C@@H](C)CO1 UVYVLBIGDKGWPX-KUAJCENISA-N 0.000 description 1
- UVYVLBIGDKGWPX-UHFFFAOYSA-N digitonine Natural products CC1C(C2(CCC3C4(C)CC(O)C(OC5C(C(O)C(OC6C(C(OC7C(C(O)C(O)CO7)O)C(O)C(CO)O6)OC6C(C(OC7C(C(O)C(O)C(CO)O7)O)C(O)C(CO)O6)O)C(CO)O5)O)CC4CCC3C2C2O)C)C2OC11CCC(C)CO1 UVYVLBIGDKGWPX-UHFFFAOYSA-N 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- YQRRQZASZVDIJJ-UHFFFAOYSA-N dimethyl(propyl)azanium;propane-1-sulfonate Chemical compound CCC[NH+](C)C.CCCS([O-])(=O)=O YQRRQZASZVDIJJ-UHFFFAOYSA-N 0.000 description 1
- 239000012972 dimethylethanolamine Substances 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 229940079868 disodium laureth sulfosuccinate Drugs 0.000 description 1
- YGAXLGGEEQLLKV-UHFFFAOYSA-L disodium;4-dodecoxy-4-oxo-2-sulfonatobutanoate Chemical compound [Na+].[Na+].CCCCCCCCCCCCOC(=O)CC(C([O-])=O)S([O-])(=O)=O YGAXLGGEEQLLKV-UHFFFAOYSA-L 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- SYELZBGXAIXKHU-UHFFFAOYSA-N dodecyldimethylamine N-oxide Chemical compound CCCCCCCCCCCC[N+](C)(C)[O-] SYELZBGXAIXKHU-UHFFFAOYSA-N 0.000 description 1
- 238000011143 downstream manufacturing Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000009144 enzymatic modification Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-N ethanesulfonic acid Chemical compound CCS(O)(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-N 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 108010091897 factor V Leiden Proteins 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000002657 fibrous material Substances 0.000 description 1
- 239000010408 film Substances 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000003365 glass fiber Substances 0.000 description 1
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 1
- 150000004820 halides Chemical class 0.000 description 1
- 229940109738 hematin Drugs 0.000 description 1
- BTIJJDXEELBZFS-QDUVMHSLSA-K hemin Chemical compound CC1=C(CCC(O)=O)C(C=C2C(CCC(O)=O)=C(C)\C(N2[Fe](Cl)N23)=C\4)=N\C1=C/C2=C(C)C(C=C)=C3\C=C/1C(C)=C(C=C)C/4=N\1 BTIJJDXEELBZFS-QDUVMHSLSA-K 0.000 description 1
- 229940025294 hemin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000140 heteropolymer Polymers 0.000 description 1
- 239000008240 homogeneous mixture Substances 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 1
- 239000004021 humic acid Substances 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- RNYJXPUAFDFIQJ-UHFFFAOYSA-N hydron;octadecan-1-amine;chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCCCC[NH3+] RNYJXPUAFDFIQJ-UHFFFAOYSA-N 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910052809 inorganic oxide Inorganic materials 0.000 description 1
- 239000010954 inorganic particle Substances 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 159000000014 iron salts Chemical class 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 150000002605 large molecules Chemical class 0.000 description 1
- 238000000608 laser ablation Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000003658 microfiber Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 108010009719 mutanolysin Proteins 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 210000000633 nuclear envelope Anatomy 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- UYDLBVPAAFVANX-UHFFFAOYSA-N octylphenoxy polyethoxyethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=C(OCCOCCOCCOCCO)C=C1 UYDLBVPAAFVANX-UHFFFAOYSA-N 0.000 description 1
- 125000001117 oleyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])/C([H])=C([H])\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000011146 organic particle Substances 0.000 description 1
- 239000000123 paper Substances 0.000 description 1
- 239000011236 particulate material Substances 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920001992 poloxamer 407 Polymers 0.000 description 1
- 229920001464 poly(sodium 4-styrenesulfonate) Polymers 0.000 description 1
- 229920000172 poly(styrenesulfonic acid) Polymers 0.000 description 1
- 239000004584 polyacrylic acid Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229940005642 polystyrene sulfonic acid Drugs 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 210000004777 protein coat Anatomy 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 229940075560 sodium lauryl sulfoacetate Drugs 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- KKDONKAYVYTWGY-UHFFFAOYSA-M sodium;2-(methylamino)ethanesulfonate Chemical compound [Na+].CNCCS([O-])(=O)=O KKDONKAYVYTWGY-UHFFFAOYSA-M 0.000 description 1
- UAJTZZNRJCKXJN-UHFFFAOYSA-M sodium;2-dodecoxy-2-oxoethanesulfonate Chemical compound [Na+].CCCCCCCCCCCCOC(=O)CS([O-])(=O)=O UAJTZZNRJCKXJN-UHFFFAOYSA-M 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000012421 spiking Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- PXQLVRUNWNTZOS-UHFFFAOYSA-N sulfanyl Chemical class [SH] PXQLVRUNWNTZOS-UHFFFAOYSA-N 0.000 description 1
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 108091035539 telomere Proteins 0.000 description 1
- 102000055501 telomere Human genes 0.000 description 1
- 210000003411 telomere Anatomy 0.000 description 1
- 229920001897 terpolymer Polymers 0.000 description 1
- 229920006029 tetra-polymer Polymers 0.000 description 1
- 238000005382 thermal cycling Methods 0.000 description 1
- 238000002145 thermally induced phase separation Methods 0.000 description 1
- 239000004416 thermosoftening plastic Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229910001428 transition metal ion Inorganic materials 0.000 description 1
- 229940072029 trilaureth-4 phosphate Drugs 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 229940046009 vitamin E Drugs 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 1
- 239000004711 α-olefin Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides methods and kits for isolating nucleic acid from a sample, preferably from a biological sample, using a microfluidic device and sedimenting reagent.
Description
METHODS FOR NUCLEIC ACID ISOLATION AND KITS USING SOLID PHASE
MATERIAL
BACKGROUND
The isolation and purification of nucleic acids (DNA and RNA, for example) from complex matrices such as blood, tissue samples, bacterial cell culture media, and forensic samples is an important process in genetic research, nucleic acid probe diagnostics, forensic DNA testing, and other areas that require amplification of the nucleic acids. A
variety of methods of preparing nucleic acids for amplification procedures are known in to the art; however, each has its limitations.
The most common method for isolating DNA from whole blood involves the isolation of peripheral blood mononuclear cells (PBMC's) using density gradients. While this method works for research applications, it is generally not suitable for use in a conventional integrated, high throughput microfluidic device.
Hypotonic buffers containing a nonionic detergent can be used to lyse red blood cells (RBC's) as well as white blood cells (WBC's) while leaving the nuclei in tact (i.e., unbroken). In another procedure, only RBC's are lysed when whole blood is subjected to freezing and thawing. The in-tact WBC's or their nuclei can be recovered by centrifugation. For lysis of RBC's without destruction of WBC's, one can also use aqueous dilution as a method. Other methods for selective lysis of RBC's include the use of ammonium chloride or quaternary ammonium salts as well as subjecting RBC's to hypotonic shock in the presence of a hypotonic buffer. However, in conventional methods using one of these approaches, substances that inhibit PCR (e.g., inhibitors of enzymes) are coprecipitated with the nuclei and/or nucleic acid. These inhibitors have to be removed prior to analysis in a conventional high throughput microfluidic device.
While treatment such as boiling, hydrolysis with proteinases, exposure to ultrasonic waves, detergents, or strong bases have been used for the extraction of DNA, alkaline extraction is among the simplest of strategies. For example, U.S. Pat. No.
5,620,852 (Lin et al.) describes an efficient extraction of DNA from whole blood performed with alkaline treatment (e.g., NaOH) at room temperature in a time frame as short as 1 minute.
However, in order to get clean DNA, removal of hemoglobin as well as plasma proteins is necessary. This has been accomplished by the use of a brief washing step, for example, by suspension of the blood in water followed by centrifugation, discarding of the supernatant, and then extraction of the pellet with NaOH (see, e.g., Biotechniques, Vol.
25, No. 4 (1998) page 588). The large volume of water used to lyse the cells makes the method unsuitable for use in standard microfluidic devices.
U.S. Patent No. 5,010,183 (Kellogg et al.) describes a centrifugal microfluidics-based platform that uses alkaline lysis for DNA extraction from blood. This method involves mixing a raw sample (e.g., 5 microliters (pL) of whole blood or an E.
Coli suspension) with 5 p.L of 10 millimolar (mM) NaOH, heating to 95°C for 1-2 minutes to lyse cells, releasing DNA and denaturing proteins inhibitory to PCR, neutralizing of the lysate by mixing with 5 p,L of 16 mM TRIS-HCl (pH 7.5), mixing the neutralized lysate with 8-10 ~.L of liquid PCR reagents and primers, followed by thermal cycling.
Unfortunately, while the reagent volumes are small and suitable for a microfluidic device, downstream processing of DNA in a microfluidic device is challenging.
Another conventional method uses a phenol chloroform extraction. However, this requires the use of toxic and corrosive chemicals and is not easily automated.
Solid phase extraction has also been used for nucleic acid isolation. For example, one method for isolating nucleic acids from a nucleic acid source involves mixing a suspension of silica particles with a buffered chaotropic agent, such as guanidinium thiocyanate, in a reaction vessel followed by addition of the sample. In the presence of the chaotrope, the nucleic acids are adsorbed onto the silica, which is separated from the liquid phase by centrifugation, washed with an alcohol water mix, and finally eluted using a dilute aqueous buffer. Silica solid phase extraction requires the use of the alcohol wash step to remove residual chaotrope without eluting the nucleic acid; however, great care must be taken to remove all traces of the alcohol (by heat evaporation or washing with another very volatile and flammable solvent) in order to prevent inhibition of sensitive enzymes used to amplify or modify the nucleic acid in subsequent steps. The nucleic acid is then eluted with water or an elution buffer. This bind, rinse, and elute procedure is the basis of many commercial kits, such as Qiagen (Valencia, CA); however, this procedure is very cumbersome and includes multiple wash steps, making it difficult to adapt to a microfluidic setting.
MATERIAL
BACKGROUND
The isolation and purification of nucleic acids (DNA and RNA, for example) from complex matrices such as blood, tissue samples, bacterial cell culture media, and forensic samples is an important process in genetic research, nucleic acid probe diagnostics, forensic DNA testing, and other areas that require amplification of the nucleic acids. A
variety of methods of preparing nucleic acids for amplification procedures are known in to the art; however, each has its limitations.
The most common method for isolating DNA from whole blood involves the isolation of peripheral blood mononuclear cells (PBMC's) using density gradients. While this method works for research applications, it is generally not suitable for use in a conventional integrated, high throughput microfluidic device.
Hypotonic buffers containing a nonionic detergent can be used to lyse red blood cells (RBC's) as well as white blood cells (WBC's) while leaving the nuclei in tact (i.e., unbroken). In another procedure, only RBC's are lysed when whole blood is subjected to freezing and thawing. The in-tact WBC's or their nuclei can be recovered by centrifugation. For lysis of RBC's without destruction of WBC's, one can also use aqueous dilution as a method. Other methods for selective lysis of RBC's include the use of ammonium chloride or quaternary ammonium salts as well as subjecting RBC's to hypotonic shock in the presence of a hypotonic buffer. However, in conventional methods using one of these approaches, substances that inhibit PCR (e.g., inhibitors of enzymes) are coprecipitated with the nuclei and/or nucleic acid. These inhibitors have to be removed prior to analysis in a conventional high throughput microfluidic device.
While treatment such as boiling, hydrolysis with proteinases, exposure to ultrasonic waves, detergents, or strong bases have been used for the extraction of DNA, alkaline extraction is among the simplest of strategies. For example, U.S. Pat. No.
5,620,852 (Lin et al.) describes an efficient extraction of DNA from whole blood performed with alkaline treatment (e.g., NaOH) at room temperature in a time frame as short as 1 minute.
However, in order to get clean DNA, removal of hemoglobin as well as plasma proteins is necessary. This has been accomplished by the use of a brief washing step, for example, by suspension of the blood in water followed by centrifugation, discarding of the supernatant, and then extraction of the pellet with NaOH (see, e.g., Biotechniques, Vol.
25, No. 4 (1998) page 588). The large volume of water used to lyse the cells makes the method unsuitable for use in standard microfluidic devices.
U.S. Patent No. 5,010,183 (Kellogg et al.) describes a centrifugal microfluidics-based platform that uses alkaline lysis for DNA extraction from blood. This method involves mixing a raw sample (e.g., 5 microliters (pL) of whole blood or an E.
Coli suspension) with 5 p.L of 10 millimolar (mM) NaOH, heating to 95°C for 1-2 minutes to lyse cells, releasing DNA and denaturing proteins inhibitory to PCR, neutralizing of the lysate by mixing with 5 p,L of 16 mM TRIS-HCl (pH 7.5), mixing the neutralized lysate with 8-10 ~.L of liquid PCR reagents and primers, followed by thermal cycling.
Unfortunately, while the reagent volumes are small and suitable for a microfluidic device, downstream processing of DNA in a microfluidic device is challenging.
Another conventional method uses a phenol chloroform extraction. However, this requires the use of toxic and corrosive chemicals and is not easily automated.
Solid phase extraction has also been used for nucleic acid isolation. For example, one method for isolating nucleic acids from a nucleic acid source involves mixing a suspension of silica particles with a buffered chaotropic agent, such as guanidinium thiocyanate, in a reaction vessel followed by addition of the sample. In the presence of the chaotrope, the nucleic acids are adsorbed onto the silica, which is separated from the liquid phase by centrifugation, washed with an alcohol water mix, and finally eluted using a dilute aqueous buffer. Silica solid phase extraction requires the use of the alcohol wash step to remove residual chaotrope without eluting the nucleic acid; however, great care must be taken to remove all traces of the alcohol (by heat evaporation or washing with another very volatile and flammable solvent) in order to prevent inhibition of sensitive enzymes used to amplify or modify the nucleic acid in subsequent steps. The nucleic acid is then eluted with water or an elution buffer. This bind, rinse, and elute procedure is the basis of many commercial kits, such as Qiagen (Valencia, CA); however, this procedure is very cumbersome and includes multiple wash steps, making it difficult to adapt to a microfluidic setting.
Ion exchange methods produce high quality nucleic acids. However, ion exchange methods result in the presence of high levels of salts that typically must be removed before the nucleic acids can be further utilized.
International Publication No. WO 01/37291 A1 (MagNA Pure) describes the use of magnetic glass particles and an isolation method in which samples are lysed by incubation with a special buffer containing a chaotropic salt and proteinase K. Glass magnetic particles are added and total nucleic acids contained in the sample are bound to their surface. Unbound substances are removed by several washing steps. Finally, purified total nucleic acid is eluted with a low salt buffer at high temperature.
Yet another conventional method involves applying a biological sample to a hydrophobic organic polymeric solid phase to selectively trap nucleic acid and subsequently remove the trapped nucleic acid with a nonionic surfactant.
Another method involves treating a hydrophobic organic polymeric material with a nonionic surfactant, washing the surface, and subsequently contacting the treated solid organic polymeric material with a biological sample to reduce the amount of nucleic acid that binds to the organic polymeric solid phase. Although these solid phase methods are effective methods for isolating nucleic acid from biological samples, other methods are needed, particularly methods that are suitable for use in microfluidic devices.
The discussion of prior publications and other prior knowledge does not constitute an admission that such material was published, known, or part of the common general knowledge.
SUMMARY
The present invention provides methods for the isolation, and preferably purification and recovery, of nucleic acids.
Nucleic acids isolated according to the invention, will be useful, for example, in assays for detection of the presence of a particular nucleic acid in a sample.
Such assays are important in the prediction and diagnosis of disease, forensic medicine, epidemiology, and public health. For example, isolated DNA may be subjected to hybridization and/or amplification to detect the presence of an infectious virus or a mutant gene in an individual, allowing determination of the probability that the individual will suffer from a disease of infectious or genetic origin. The ability to detect an infectious virus or a mutation in one sample among the hundreds or thousands of samples being screened takes on substantial importance in the early diagnosis or epidemiology of an at-risk population for disease, e.g., the early detection of HIV infection, cancer or susceptibility to cancer, or in the screening of newborns for diseases, where early detection may be instrumental in diagnosis and treatment. In addition, the methods of the present invention can also be used in basic research laboratories to isolate nucleic acid from cultured cells or biochemical reactions. The nucleic acid can be used for enzymatic modification such as restriction enzyme digestion, sequencing, and amplification.
1o The present invention provides methods and kits for isolating nucleic acid from a sample that includes nucleic acid (e.g., DNA, RNA, PNA), which may or may not be included within nuclei-containing cells (e.g., white blood cells). These methods involve ultimately separating nucleic acid from inhibitors, such as heme and degradation products thereof (e.g., iron ions or salts thereof), which are undesirable because they can inhibit amplification reactions (e.g., as are used in PCR reactions).
Certain embodiments of the invention involve retaining inhibitors in or on a solid phase material (i.e., adhering the inhibitors to the material) without retaining a significant amount of nucleic acid. Suitable solid phase materials typically .include a solid matrix in any form (e.g., particles, fibrils, a membrane) with capture sites (e.g., chelating functional groups) attached thereto, a coating reagent (preferably, a surfactant) coated on the solid phase material, or both.
In one embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method including: providing a sample including nucleic acid-containing material and inhibitors (typically, contained in cells; such nucleic acid-containing material and cells containing inhibitors may be the same or different); if the sample includes cells containing inhibitors, the method includes optionally contacting the sample with a first lysing reagent under conditions effective to break cell merribranes and release inhibitors and form a lysed sample including nucleic acid-containing material and inhibitors; forming a concentrated region of the sample (typically, lysed sample); wherein the concentrated region of the sample (typically, lysed sample) includes nucleic acid-containing material and inhibitors; substantially separating the concentrated region from a less concentrated region of the sample (typically, lysed sample); contacting the separated concentrated region of the sample (typically, lysed sample) with a solid phase material to preferentially adhere at least a portion of the inhibitors (i.e., at least a portion of at least one inhibitor) to the solid phase material, wherein the solid phase material includes a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barner, and combinations thereof; optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material;
and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method including: providing a sample including cells containing inhibitors and cells containing nuclei (such cells containing inhibitors and cells containing nuclei may be the same or different); contacting the biological sample with a nonionic surfactant under conditions effective to break cell membranes and release nuclei and inhibitors and form a lysed sample; forming a concentrated region of the lysed sample;
wherein the concentrated region of the sample includes nuclei and inhibitors;
substantially separating the concentrated region of the lysed sample from the less concentrated region of the sample; contacting the separated concentrated region of the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material includes a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof; optionally further lysing the nuclei to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nucleic acid-containing and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method including: providing a microfluidic device including a loading chamber, a valued process chamber, and a separation chamber including a solid phase material; providing a sample including nucleic acid-containing material and inhibitors (typically, contained in cells; such nucleic acid-containing material and cells containing inhibitors may be the same or different); placing the sample in the loading chamber; if the sample includes cells containing inhibitors, optionally contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample including nucleic acid-containing material and inhibitors; transfernng the sample (typically, lysed sample) to a valued process chamber; forming a concentrated region of the sample (typically, lysed sample) in the valued process chamber, wherein the concentrated region of the sample (typically, lysed sample) includes nucleic acid-containing material and inhibitors;
substantially separating the concentrated region from a less concentrated region of the sample (typically, lysed sample); transferring the separated concentrated region of the sample (typically, lysed sample) to the separation chamber for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material includes capture sites (e.g., chelating functional groups), a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof; optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material;
and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method including: providing a microfluidic device including a loading chamber, a valued process chamber, and a separation chamber including a solid phase material; providing a sample including cells containing inhibitors and cells containing nuclei (such cells containing inhibitors and cells containing nuclei may be the same or different); placing the sample in the loading chamber;
contacting the sample with a nonionic surfactant under conditions effective to break cell membranes and release nuclei and inhibitors to form a lysed sample; transferring the lysed sample to a valued process chamber; forming a concentrated region of the lysed sample in the valued process chamber, wherein the concentrated region of the lysed sample includes nuclei and inhibitors; substantially separating the concentrated region from a less concentrated region of the lysed sample; transferring the separated concentrated region of the lysed sample to the separation chamber for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material includes capture sites (e.g., chelating functional groups), a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof; optionally further lysing the nuclei to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the i5 nuclei and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method including: providing a sample including nucleic acid and inhibitors; contacting the sample with a solid phase material to preferentially 2o adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material includes a polytetrafluoroethylene fibril matrix, soiptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barner, and combinations thereof;
25 optionally lysing nucleic acid-containing material, if present, to release nucleic acid before, simultaneous with, or after contacting the lysed sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic 30 surfactant.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method including: providing a sample including nucleic acid-containing material (e.g., nuclei) and inhibitors (typically, contained in cells; such nucleic acid-containing material and cells containing inhibitors may be the same or different); if the sample includes cells containing inhibitors, optionally contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample including nucleic acid-containing material and inhibitors; contacting the sample (typically, lysed sample) with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material includes a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof (preferably, a surfactant); optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the lysed sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic surfactant.
2o In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method including: providing a sample including nucleic acid-containing material (e.g., nuclei) and cells containing inhibitors (which may be the same or different); contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and for~rr a lysed sample including nucleic acid-containing material and inhibitors; contacting the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material includes a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, 3o a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof (preferably, a surfactant); separating at least a portion of the nucleic acid-containing material from the solid phase material having at least a portion of the inhibitors adhered thereto; and after separating the nucleic acid-containing material, further lysing the nucleic acid-containing material to release nucleic acid.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method including: providing a microfluidic device including a loading chamber and a separation chamber including a solid phase material;
providing a sample including nucleic acid-containing material and inhibitors (typically, contained in cells; such nucleic acid-containing material and cells containing inhibitors may be the same or different); placing the sample in the loading chamber; if the sample includes cells containing inhibitors, optionally contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample including nucleic acid-containing material and inhibitors;
transferring the sample (typically, lysed sample) to the separation chamber to contact the solid phase material and to preferentially adhere at least a portion of the inhibitors to the solid phase material; optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the sample with the solid phase material; wherein the solid phase material includes capture sites (e.g., chelating functional groups), a coating reagent coated on the solid phase material, or both;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barner, and combinations thereof; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic surfactant.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method including: providing a microfluidic device including a loading chamber and a separation chamber including a solid phase material;
providing a sample including nucleic acid-containing material and cells containing inhibitors (which may be the same or different); placing the sample in the loading 3o chamber; contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample including nucleic acid-containing material and inhibitors; transferring the lysed sample to the separation chamber to contact the solid phase material and to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material includes capture sites (e.g., chelating functional groups), a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barner, and combinations thereof; and separating at least a portion of the nucleic acid-containing material from the solid phase material having at least a portion of the inhibitors adhered thereto; and after separating the nucleic acid-containing material, further lysing the nucleic l0 acid-containing material to release nucleic acid.
The present invention also provides kits for carrying out the various methods of the present invention.
DEFINITIONS
"Nucleic acid" shall have the meaning known in the art and refers .to DNA
(e.g., genomic DNA, cDNA, or plasmid DNA), RNA (e.g., mRNA, tRNA, or rRNA), and PNA.
It can be in a wide variety of forms, including, without limitation, double-stranded or single-stranded configurations, circular form, plasmids, relatively short oligonucleotides, peptide nucleic acids also called PNA's (as described in Nielsen et al., Chem.
Soc. Rev., 26, 73-78 (1997)), and the like. The nucleic acid can be genomic DNA, which can include an entire chromosome or a portion of a chromosome. The DNA can include coding (e.g., for coding mRNA, tRNA, and/or rRNA) and/or noncoding sequences (e.g., centromeres, telomeres, intergeriic regions, introns, transposons, and/or microsatellite sequences). The nucleic acid can include any of the naturally occurnng nucleotides as well as artificial or chemically modified nucleotides, mutated nucleotides, etc. The nucleic acid can include a non-nucleic acid component, e.g., peptides (as in PNA's), labels (radioactive isotopes or fluorescent markers), and the like.
"Nucleic acid-containing material" refers to a source of nucleic acid such as a cell (e.g., white blood cell, enucleated red blood cell), a nuclei, or a virus, or any other composition that houses a structure that includes nucleic acid (e.g., plasmid, cosmid, or viroid, archeobacteriae). The cells can be prokaryotic (e.g., gram positive or gram negative bacteria) or eukaryotic (e.g., blood cell or tissue cell). If the nucleic acid-containing material is a virus, it can include an RNA or a DNA genome; it can be virulent, attenuated, or noninfectious; and it can infect prokaryotic or eukaryotic cells. The nucleic acid-containing material can be naturally occurnng, artificially modified, or artificially created.
"Isolated" refers to nucleic acid (or nucleic acid-containing material) that has been separated from at least a portion of the inhibitors (i.e., at least a portion of at least one inhibitor) in a sample. This includes separating desired nucleic acid from other materials, e.g., cellular components such as proteins, lipids, salts, and other inhibitors. More 1o preferably, the isolated nucleic acid is substantially purified.
"Substantially purified"
refers to isolating nucleic acid of at least 3 picogram per microliter (pg/p,L), preferably at least 2 nanogram/microliter (ng/~,L), and more preferably at least 15 ng/p,L, while reducing the inhibitor amount from the original sample by at least 20%, preferably by at least 80%
and more preferably by at least 99%. The contaminants are typically cellular components and nuclear components such as heme and related products (hemin, hematin) and metal ions, proteins, lipids, salts, etc., other than the solvent in the sample.
Thus, the term "substantially purified" generally refers to separation of a majority of inhibitors (e.g., heme and it degradation products) from the sample, so that compounds capable of interfering with the subsequent use of the isolated nucleic acid are at least partially removed.
"Adheres to" or "adherence" or "binding" refer to reversible retention via a wide variety of mechanisms, including weak forces such as Van der Waals interactions, electrostatic interactions, affinity binding, or physical trapping. The use of this term does not imply a mechanism of action, and includes adsorptive and absorptive mechanisms.
"Solid phase material" refers to an inorganic and/or organic material, preferably a polymer made of repeating units, which may be the same or different, of organic and/or inorganic compounds of natural and/or synthetic origin. This includes homopolymers and heteropolymers (e.g., copolymers, terpolymers, tetrapolymers, etc., which may be random or block, for example). This term includes fibrous or particulate forms of a material, which can be readily prepared by methods well-known in the art. Such materials typically form a porous matrix, although for certain embodiments, the solid phase also refers to a solid surface, such as a nonporous sheet of polymeric material.
The solid phase material may include capture sites. "Capture sites" refer to sites on the solid phase material to which a material adheres. Typically, the capture sites include functional groups or molecules that are either covalently attached or otherwise attached (e.g., hydrophobically attached) to the solid phase material.
The phrase "coating reagent coated on the solid phase material" refers to a material coated on at least a portion of the solid phase material, e.g., on at least a portion of the fibril matrix and/or sorptive particles.
"Surfactant" refers to a substance that lowers the surface or interfacial tension of the medium in which it is dissolved.
"Strong base" refers to a base that is completely dissociated in water, e.g., NaOH.
"Polyelectrolyte" refers to an electrolyte that, is a charged polymer, typically of relatively high molecular weight, e.g., polystyrene sulfonic acid.
"Selectively permeable polymeric barrier" refers to a polymeric barrier that allows for selective transport of a fluid based on size and charge.
"Concentrated region" refers to a region of a sample that has a higher concentration of nucleic acid-containing material, nuclei, and/or nucleic acid, which can be in a pellet form, relative to the less concentrated region.
"Substantially separating" as used herein, particularly in the context of separating a concentrated region of a sample from a less concentrated region of a sample, means 2o removing at least 40% of the total amount of nucleic acid (whether it be free, within nuclei, or within other nucleic acid-containing material) in less than 25% of the total volume of the sample. Preferably, at least 75% of the total amount of nucleic acid in less than 10% of the total volume of sample is separated from the remainder of the sample.
More preferably, at least 95% of the total amount of nucleic acid in less than 5% of the total volume of sample is separated from the remainder of the sample.
"Inhibitors" refer to inhibitors of enzymes used in amplification reactions, for example. Examples of such inhibitors typically include iron ions or salts thereof (e.g., Fe2+ or salts thereof) and other metal salts (e.g., alkali metal ions, transition metal ions).
Other inhibitors can include proteins, peptides, lipids, carbohydrates, heme and its 3o degradation products, urea, bile acids, humic acids, polysaccharides, cell membranes, and cytosolic components. The major inhibitors in human blood for PCR are hemoglobin, lactofernn, and IgG, which are present in erythrocytes, leukocytes, and plasma, respectively. The methods of the present invention separate at least a portion of the inhibitors (i.e., at least a portion of at least one type of inhibitor) from nucleic acid-containing material. As discussed herein, cells containing inhibitors can be the same as the cells containing nuclei or other nucleic acid-containing material.
Inhibitors can be contained in cells or be extracellular. Extracellular inhibitors include all inhibitors not contained within cells, which includes those inhibitors present in serum or viruses, for example.
"Preferentially adhere at least a portion of the inhibitors to the solid phase material"
1o means that one or more types of inhibitors will adhere to the solid phase material to a greater extent than nucleic acid-containing material (e.g., nuclei) and/or nucleic acid, and typically without adhering a substantial portion of the nucleic acid-containing material and/or nuclei to the solid phase material.
"Microfluidic" refers to a device with one or more fluid passages, chambers, or 15 conduits that have at least one internal cross-sectional dimension, e.g., depth, width, length, diameter, etc., that is less than 500 pm, and typically between 0.1 p,m and 500 p,m.
In the devices used in the present invention, the microscale channels or chambers preferably have at least one cross-sectional dimension between 0.1 p,m and 200 p,m, more preferably between 0.1 pm and 100 pm, and often between 1 p,m and 20 p.m.
Typically, a 2o microfluidic device includes a plurality of chambers (process chambers, separation chambers, mixing chambers, waste chambers, diluting reagent chambers, amplification reaction chambers, loading chambers, and the like), each of the chambers defining a volume for containing a sample; and at least one distribution channel connecting the plurality of chambers of the array; wherein at least one of the chambers within the array 25 can include a solid phase material (thereby often being referred to as a separation chamber) and/or at least one of the process chambers within the array can include a lysing reagent (thereby often being referred to as a mixing chamber), for example.
The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.
As used herein "a " "an " "the " "at least one " and "one or more" are used , , , , , interchangeably and mean one or more.
Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
Furthermore, various embodiments are described in which the various elements of each embodiment could be used in other embodiments, even though not specifically described.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1-2 are representations of microfluidic devices used in certain methods of the present invention.
2o DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention provides various methods and kits for isolating nucleic acid from a sample, typically a biological sample, preferably in a substantially purified form.
The present invention provides methods and kits for isolating nucleic acid from a sample that includes nucleic acid (e.g., DNA, RNA, PNA), which may or may not be included within nuclei-containing cells (e.g., white blood cells).
It should be understood that although the methods are directed to isolating nucleic acid from a sample, the methods do not necessarily remove the nucleic acid from the nucleic acid-containing material (e.g., nuclei). That is, further steps may be required to further separate the nucleic acid from the nuclei, for example.
The methods of the present invention involve ultimately separating nucleic acid from inhibitors, such as heme and degradation products thereof (e.g., iron salts), which are undesirable because they can inhibit amplification reactions (e.g., as are used in PCR
reactions). More specifically, the methods of the present invention involve separating at least a portion of the nucleic acid in a sample from at least a portion of at least one type of inhibitor. Preferred methods involve removing substantially all the inhibitors in a sample containing nucleic acid such that the nucleic acid is substantially pure. For example, the final concentration of iron-containing inhibitors is no greater than about 0.8 micromolar (p,M), which is the current level tolerated in conventional PCR systems.
In order to get clean DNA from whole blood, removal of hemoglobin as well as plasma proteins is typically desired. When red blood cells are lysed, heme and related to compounds are released that inhibit Taq Polymerise. The normal hemoglobin concentration in whole blood is 15 grams (g) per 100 milliliters (mL) based on which the concentration of heme in hemolysed whole blood is around 10 millimolar (mM).
For PCR
to work out satisfactorily, the concentration of heme should be reduced to the micromolar (pM) level. This can be achieved by dilution or by removal of inhibitors using a material that binds inhibitors, for example.
Typically, a sample containing nucleic acid is processed in a flow-through receptacle, although this receptacle is not a necessary requirement of the present invention.
Preferably, for certain methods of the present invention, the processing equipment is in a microfluidic format.
SAMPLES
The methods of the present invention can be used to isolate nucleic acids from a wide variety of samples, particularly biological samples, such as body fluids (e.g., whole blood, blood serum, urine, saliva, cerebral spinal fluid, semen, or synovial lymphatic fluid), various tissues (e.g., skin, hair, fur, feces, tumors, or organs such as liver or spleen), cell cultures or cell culture supernatants, etc. The sample can be a food sample, a beverage sample, a fermentation broth, a clinical sample used to diagnose, treat, monitor, or cure a disease or disorder, a forensic sample, an agricultural sample (e.g., from a plant or animal), or an environmental sample (e.g., soil, dirt, or garbage).
3o Biological samples are those of biological or biochemical origin. Those suitable for use in the methods of the present invention can be derived from mammalian, plant, bacterial, or yeast sources. The biological sample can be in the form of single cells or in the form of a tissue. Cells or tissue can be derived from in vitro culture.
Significantly, certain embodiments of the invention use whole blood without any preprocessing (e.g., lysing, filtering, etc.) as the sample of interest.
For certain embodiments, a sample such as whole blood can be preprocessed by centrifuging and the white blood cells (i.e., the huffy coat) separated from the blood and used as the sample in the methods of the invention.
For certain embodiments, a sample can be subjected to ultracentrifugation to concentrate the sample prior to subjecting it to a process of the present invention.
to The sample can be a solid sample (e.g., solid tissue) that is dissolved or dispersed in water or an organic medium, or from which the nucleic acid has been extracted into water or an organic medium. For example, the sample can be an organ homogenate (e.g., liver, spleen). Thus, the sample can include previously extracted nucleic acid (particularly if it is a solid sample).
The type of sample is not a limitation of the present invention. Typically, however, the sample will include nucleic acid-containing material and inhibitors from which the nucleic acid needs to be separated. In this context, nucleic acid-containing material refers to cells (e.g., white blood cell; bacterial cells), nuclei, viruses, or any other composition that houses a structure that includes nucleic acid (e.g., plasmid, cosmid, or viroid, archeobacteriae). In certain preferred embodiments of such methods, the nucleic acid-containing material includes nuclei. In certain embodiments, such nuclei are in tact (i.e., substantially unlysed) when they contact the solid phase material described herein.
1n certain embodiments, the sample may be partially lysed (e.g., pre-lysed to release inhibitors), in which case lysing may be required in the process of the present invention, or fully lysed. Thus, a sample can include free (e.g., not within cells) nucleic acid and free (e.g., not within cells) inhibitors.
The isolated (i.e., separated from inhibitors) nucleic acid can be used, preferably without further purification or washing, for a wide variety of applications (e.g., amplification, sequencing, labeling, annealing, restriction digest, ligation, reverse 3o transcriptase, hybridization, Southern blot, Northern blot, etc.). In particularly, it can be used for determining a subject's genome. It can be used for the diagnosis of the presence of a microorganism (e.g., bacteria, virus) in a sample, and subsequently can be used for monitoring and/or remedying the damage caused by the microorganism to the source of the sample. The methods, materials, systems, and kits of the present invention are especially well-suited for preparing nucleic acid extracts for use in amplification techniques (e.g., PCR, LCR, MASBA, SDA, and bDNA) used in high throughput or automated processes, particularly microfluidic systems. Thus, for certain embodiments of the present invention, the isolated nucleic acid is transferred to an amplification reaction chamber (such as a PCR
sample chamber in a microfluidic device).
The nucleic acids may be isolated (i.e., separated from inhibitors) according to the 1o invention from an impure, partially pure, or a pure sample. The purity of the original sample is not critical, as nucleic acid may be isolated from even grossly impure samples.
For example, nucleic acid may be obtained from an impure sample of a biological fluid such as blood, saliva, or tissue. If an original sample of higher purity is desired, the sample may be treated according to any conventional means known to those of skill in the art prior to undergoing the methods of the present invention. For example, the sample may be processed so as to remove certain impurities such as insoluble materials prior to subjecting the sample to a method of the present invention.
The nucleic acid isolated as described herein may be of any molecular weight and in single-stranded form, double-stranded form, circular, plasmid, etc. Various types of 2o nucleic acid can be separated from each other (e.g., RNA from DNA, or double-stranded DNA from single-stranded DNA). For example, small oligonucleotides or nucleic acid molecules of about 10 to about 50 bases in length, much longer molecules of about 1000 bases to about 10,000 bases in length, and even high molecular weight nucleic acids of about 50 kb to about 500 kb can be isolated using the methods of the present invention. In some aspects, a nucleic acid isolated according to the invention may preferably be in the range of about 10 bases to about 100 kilobases.
The nucleic acid-containing sample may be in a wide variety of volumes. For example, the applied volume may be as large as 1 liter or as small as 1 ~L, or even less.
The sample size typically varies depending on the equipment used to carry out the method.
For a microfluidic format, typically very small volumes, e.g., 10 p.L (and preferably, no greater than 100 p.L) are preferred. It should be understood that larger samples can be used if preprocessed, such as by concentrating.
For low copy number genes, one typically would need a larger sample size to ensure that the sequence of interest is present in the sample. Larger sample sizes, however, have a greater amount of inhibitors and do not typically lend themselves to a microfluidic format. Thus, for a low copy number situation, it may be necessary to use a 100 p,L or higher volume in order to get a reproducible result; however, the number of samples processed per microfluidic device may be reduced due to the higher sample volume.
l0 In the methods of the present invention, a centrifugation step to concentrate nucleic acid-containing material is useful for low copy number samples. However, while the nucleic acid concentration is increased substantially at the bottom of the process chamber, the inhibitor concentration is still high. While most of the inhibitors, the proteins in the serum and the broken RBC's (e.g., heme and heme-related products) are removed in the is supernatant; the nucleic acid-containing concentrated region of the sample still has a significant amount of inhibitor present; however, the ratio of nucleic acid to that of the inhibitor is very high, resulting in an enriched sample with respect to nucleic acid. This concentrated region of the sample can then be contacted with a solid phase material, as described herein, to remove residual inhibitors.
20 For high copy number genes, a sample size as small as 2 ~L can be used, but reproducibility is better with larger volumes (e.g., 20 pL). In the case of smaller volumes, higher throughputs (i.e., number of samples processed per microfluidic device) can be obtained. In the case of larger volumes (e.g., 20 pL), it may not be necessary to go through a pre-spin step for concentration of nucleic acid-containing cells.
25 For certain embodiments, the nucleic acid-containing sample applied to the solid phase material may be any amount, that amount being determined by the amount of the solid phase material. Preferably, the amount of nucleic acid in a sample applied to the solid phase material is less than the dried weight of the solid phase material, typically about 1/10,000 to about 1/100 (weight nucleic acid/solid phase). The amount of nucleic 30 acid in a sample applied to the solid phase material may be as much as 100 grams or as little as 1 picogram, for example.
The desired nucleic acid isolated from the methods of the present invention is preferably in an amount of at least 20%, more preferably in an amount of at least 30%, more preferably at least 70%, and most preferably at least 90%, of the amount of total nucleic acid in the originally applied sample. Thus, certain preferred methods of the present invention provide for high recovery of the desired nucleic acid from a sample.
Furthermore, exceedingly small amounts of nucleic acid molecules may be quantitatively recovered according to the invention. The recovery or yield is mainly dependent on the quality of the sample rather than the procedure itself. Because certain embodiments of the invention provide a nucleic acid preparation that does not require concentration from a to large volume, the invention avoids risk of loss of the nucleic acid.
Having too much DNA in a PCR sample can be detrimental to amplification of DNA as there are a lot of misprimed sites. This results in a large number of linearly or exponentially amplified non-target sequences. Since the specificity of the amplification is lost as the amount of non-target DNA is increased, the exponential accumulation of the target sequence of interest does not occur to any significant degree. Thus, it is desirable to control the amount of DNA that goes into each PCR sample. The DNA amount is typically not more than 1 microgram/reaction, typically at least 1 picogram/reaction.
The typical final DNA concentration in a PCR mixture ranges from 0.15 nanogram/microliter to 1.5 nanograms/microliter. In the case of a microfluidic device, a sample can be split after 2o clean-up, prior to PCR, such that each sample has the right amount of DNA.
Alternatively, a sample can be diluted sufficiently in a sample processing device (particularly, a microfluidic device) that includes a variable valued process chamber, described in greater detail below, so that the right amount of DNA is present in each PCR
mixture. 1n a diagnostic setting, since the amount of white blood cells can vary significantly, it is hard to apriori predict the amount of DNA that will be isolated.
However, a useful range is 3 micrograms (p.g) to 12 pg of DNA per 200 pL of blood. For buffy coats, 25 pg to 50 pg per 200 pL of buffy coat is a useful range.
LYSING REAGENTS AND CONDITIONS
3o For certain embodiments of the invention, at some point during the process, cells within the sample, particularly nucleic acid-containing cells (e.g., white blood cells, bacterial cells, viral cells) are lysed to release the contents of the cells and form a sample (i.e., a lysate). Lysis herein is the physical disruption of the membranes of the cells, refernng to the outer cell membrane and, when present, the nuclear membrane.
This can be done using standard techniques, such as by hydrolyzing with proteinases followed by heat inactivation of proteinases, treating with surfactants (e.g., nonionic surfactants or sodium dodecyl sulfate), guanidinium salts, or strong bases (e.g., NaOH), disrupting physically (e.g., with ultrasonic waves), boiling, or heating/cooling (e.g., heating to at least 55°C (typically to 95°C) and cooling to room temperature or below (typically to 8°C)), which can include a freezing/thawing process. Typically, if a lysing reagent is used, it is in to aqueous media, although organic solvents can be used, if desired.
Lysing of red blood cells (RBC's) without the destruction of white blood cells (WBC's) in whole blood can occur to release inhibitors through the use of water (i.e., aqueous dilution) as the lysing agent (i.e., lysing reagent). Alternatively, ammonium chloride or quaternary ammonium salts can also be used to break RBC's. The RBC's can also be lysed by hypotonic shock with the use of a hypotonic buffer. The in-tact WBC's or their nuclei can be recovered by centrifugation, for example.
Typically, a stronger lysing reagent, such as a surfactant, can be used to lyse RBC's as well as nucleic acid-containing cells (e.g., white blood cells (WBC's), bacterial cells, viral cells) to release inhibitors, nuclei, and/or nucleic acid. For example, a nonionic surfactant can be used to lyse RBC's as well as WBC's while leaving the nuclei in tact.
Nonionic surfactants, cationic surfactants, anionic surfactants, and zwitterionic surfactants can be used to lyse cells. Particularly useful are nonionic surfactants.
Combinations of surfactants can be used if desired. A nonionic surfactant such as TRITON X-100 can be added to a TRIS buffer containing sucrose and magnesium salts for isolation of nuclei.
The amount of surfactant used for lysing is sufficiently high to effectively lyse the sample, yet sufficiently low to avoid precipitation, for example. The concentration of surfactant used in lysing procedures is typically at least 0.1 wt-%, based on the total weight of the sample. The concentration of surfactant used in lysing procedures is typically no greater than 4.0 wt-%, and preferably, no greater than 1.0 wt-%, based on the total weight of the sample. The concentration is usually optimized in order to obtain complete lysis in the shortest possible time with the resulting mixture being PCR compatible. In fact, the nucleic acid in the formulation added to the PCR cocktail should allow for little or no inhibition of real-time PCR.
If desired, a buffer can be used in admixture with the surfactant. Typically, such buffers provide the sample with a pH of at least 7, and typically no more than 9.
Typically, an even stronger lysing reagent, such as a strong base, can be used to lyse any nuclei contained in the nucleic acid-containing cells (as in white blood cells) to release nucleic acid. For example, the method described in U.S. Pat. No.
5,620,852 (Lin et al.), which involves extraction of DNA from whole blood with alkaline treatment (e.g., NaOH) at room temperature in a time frame as short as 1 minute, can be adapted to certain l0 methods of the present invention. Generally, a wide variety of strong bases can be used to create an effective pH (e.g., 8-13, preferably 13) in an alkaline lysis procedure. The strong base is typically a hydroxide such as NaOH, LiOH, KOH; hydroxides with quaternary nitrogen-containing cations (e.g., quaternary ammonium) as well as bases such as tertiary, secondary or primary amines. Typically, the concentration of the strong base is at least 15 0.01 Normal (I~, and typically, no more than 1 N. Typically, the mixture can then be neutralized, particularly if the nucleic acid is subjected to PCR. In another procedure, .
heating can be used subsequent to lysing with base to further denature proteins followed by neutralizing the sample.
One can also use Proteinase K with heat followed by heat inactivation of proteinase 20 K at higher temperatures for isolation of nucleic acids from the nuclei or WBC.
One can also use a commercially available lysing agent and neutralization agent such as in Sigma's Extract-N-Amp Blood PCR kit scaled down to microfluidic dimensions. Stonger lysing solutions such as POWERLYSE from GenPoint (Oslo, Norway) for lysing difficult bacteria such as Staphylococcus, Streptococcus, etc., can be 25 used to advantage in certain methods of the present invention.
In another procedure, a boiling method can be used to lyse cells and nuclei, release DNA, and precipitate hemoglobin simultaneously. The DNA in the supernatant can be used directly for PCR without a concentration step, making this procedure useful for low copy number samples.
3o For infectious diseases, it may be necessary to analyze bacterial or viruses from whole blood. For example, in the case of bacteria, white blood cells may be present in conjunction with bacterial cells. In methods that use a microfluidic device, it would be possible to lyse red blood cells to release inhibitors, and then separate out bacterial cells and white blood cells by centrifugation, for example, prior to further lysing.
This concentrated slug of nucleic acid-containing cells (bacterial and white blood cells/nuclei) can be moved further into a chamber containing the solid phase material for removal of inhibitors. Then, the bacterial cells, for example, can be lysed.
Bacterial cell lysis, depending on the type, may be accomplished using heat.
Alternatively, bacterial cell lysis can occur using enzymatic methods (e.g., lysozyme, mutanolysin) or chemical methods. The bacterial cells are preferably lysed by alkaline lysis.
The use of bacteria for propagation of plasmids is common in the study of genomics, analytic molecular biology, preparatory molecular biology, etc. In the case of the bacterium containing plasmid, genetic material from both the bacterium and the plasmid are present. A clean-up procedure to separate cellular proteins and cellular fragments from genomic DNA can be carried out using a method of the present invention.
The supernatant thus obtained, which contains the plasmid DNA, is called the "cleared lysate." The cleared lysate can be further purified using a variety of means, such as anion-exchange chromatography, gel filtration, or precipitation with alcohol.
In a specific example of a protocol for bacterial cultures, which can be incorporated into a microfluidic device, an E. Coli cell culture is centrifuged and resuspended in TE
buffer (10 mM TRIS, 1 mM EDTA, pH 7.5) and lysed by the addition of 0.1 M
NaOH/1%
SDS (sodium dodecyl sulfate). The cell lysis is stopped by the addition of 1 volume of 3 M (three molar) potassium acetate (pH 4.8) and the supernatant centrifuged.
The cell lysate is further purified to get clean plasmid DNA.
Plasma and serum represent the majority of specimens submitted for molecular testing that include viruses. After fractionation of whole blood, plasma or serum samples can be used for the extraction of viruses (i.e., viral particles). For example, to isolate DNA
from viruses, it is possible in the microfluidic case, to first separate out the serum by spinning blood. By the use of the variable valve, which is described in greater detail below, the serum alone can be emptied into another chamber. The serum can then be centrifuged to concentrate the virus or can be used directly in subsequent lysis steps.after removal of the inhibitors using a solid phase material, for example, as described herein.
The solid phase material could absorb the solution such that the virus particles do not go through the material. The virus particles can then be eluted out in a small elution volume.
The virus can be lysed by heat or by enzymatic or chemical means, for example, by the use of surfactants, and used for downstream applications, such as PCR or real-time PCR. In cases where viral RNA is required, it may be necessary to have an RNAse inhibitor added to the solution to prevent degradation of RNA.
SOLID PHASE MATERIAL
For certain embodiments of the invention, it has been found that inhibitors will adhere to solid phase (preferably polymeric) materials that include a solid matrix in any form (e.g., particles, fibrils, a membrane), preferably with capture sites (e.g., chelating functional groups) attached thereto, a coating reagent (preferably, surfactant) coated on the solid phase material (i.e., at least a portion thereof), or both. The coating reagent can be a cationic, anionic, nonionic, or zwitterionic surfactant. Alternatively, the coating reagent can be a polyelectrolyte, a strong base, or a selectively permeable polymeric barner.
Various combinations of coating reagents can be used if desired.
The solid phase material useful in the methods of the present invention may include a wide variety of organic and/or inorganic materials that retain inhibitors such as heme and heme degradation products, particularly iron ions, for example. Such materials are functionalized with capture sites (preferably, chelating groups), coated with one or more coating reagents (e.g., surfactants, polyelectrolytes, or strong bases), or both.
Typically, the solid phase material includes an organic polymeric matrix.
Generally suitable materials are chemically inert, physically and chemically stable, and compatible with a variety of biological samples. Examples of solid phase materials include silica, zirconia, alumina beads, metal colloids such as gold, gold coated sheets that have been functionalized through mercapto chemistry, for example, to generate capture sites. Examples of suitable polymers include for example, polyolefins and fluorinated polymers. The solid phase material is typically washed to remove salts and other contaminants prior to use. It can either be stored dry or in aqueous suspension ready for use. The solid phase material is preferably used in a flow-through receptacle, for example, such as a pipet, syringe, or larger column, microtiter plate, or microfluidic device, although suspension methods that do not involve such receptacles could also be used.
The solid phase material useful in the methods of the present invention can include a wide variety of materials in a wide variety of forms. For example, it can be in the form of particles or beads, which may be loose or immobilized, fibers, foams, frits, microporous film, membrane, or a substrate with microreplicated surface(s). If the solid phase material includes particles, they are preferably uniform, spherical, and rigid to ensure good fluid flow characteristics.
For flow-through applications of the present invention, such materials are typically 1o in the form of a loose, porous network to allow uniform and unimpaired entry and exit of large molecules and to provide a large surface area. Preferably, for such applications, the solid phase material has a relatively high surface area, such as, for example, more than one meter squared per gram (mz/g). For applications that do not involve the use of a flow-through device, the solid phase material may or may not be in a porous matrix.
Thus, membranes can also be useful in certain methods of the present invention.
For applications that use particles or beads, they may be introduced to the sample or the sample introduced into a bed of particles/beads and removed therefrom by centrifuging, for example. Alternatively, particles/beads can be coated (e.g., pattern coated) onto an inert substrate (e.g., polycarbonate or polyethylene), optionally coated with 2o an adhesive, by a variety of methods (e.g., spray drying). If desired, the substrate can be microreplicated for increased surface area and enhanced clean-up. It can also be pretreated with oxygen plasma, e-beam or ultraviolet radiation, heat, or a corona treatment process.
This substrate can be used, for example, as a cover film, or laminated to a cover film, on a reservoir in a microfluidic device.
In one embodiment, the solid phase material includes a fibril matrix, which may or may not have particles enmeshed therein. The fibril matrix can include any of a wide variety of fibers. Typically, the fibers are insoluble in an aqueous environment. Examples include glass fibers, polyolefin fibers, particularly polypropylene and polyethylene microfibers, aramid fibers, a fluorinated polymer, particularly, polytetrafluoroethylene fibers, and natural cellulosic fibers. Mixtures of fibers can be used, which may be active or inactive toward binding of nucleic acid. Preferably, the fibril matrix forms a web that is at least about 15 microns, and no greater than about 1 millimeter, and more preferably, no greater than about 500 microns thick.
If used, the particles are typically insoluble in an aqueous environment. They can be made of one material or a combination of materials, such as in a coated particle. They can be swellable or nonswellable, although they are preferably nonswellable in water and organic liquids. Preferably, if the particle is doing the adhering, it is made of nonswelling, hydrophobic material. They can be chosen for their affinity for the nucleic acid. Examples of some water swellable particles are described in U.S. Pat. Nos. 4,565,663 (Errede et al.), 4,460,642 (Errede et al.), and 4,373,519 (Errede et al.). Particles that are nonswellable in to water are described in U.S. Pat. Nos. 4,810,381 (Hagen et al.), 4,906,378 (Hagen et al.), 4,971,736 (Hagen et al.); and 5,279,742 (Markell et al.). Preferred particles are polyolefin particles, such as polypropylene particles (e.g., powder). Mixtures of particles can be used, which may be active or inactive toward binding of nucleic acid.
If coated particles are used, the coating is preferably an aqueous- or organic-15 insoluble, nonswellable material. The coating may or may not be one to which nucleic acid will adhere. Thus, the base particle that is coated can be inorganic or organic. The base particles can include inorganic oxides such as silica, alumina, titanic, zirconia, etc., to which are covalently bonded organic groups. For example, covalently bonded organic groups such as aliphatic groups of varying chain length (C2, C4, C8, or C18 groups) can 2o be used.
Examples of suitable solid phase materials that include a fibril matrix are described in U.S. Pat. Nos. 5,279,742 (Markell et al.), 4,906,378 (Hagen et al.), 4,153,661 (Ree et al.), 5,071,610 (Hagen et al.), 5,147,539 (Hagen et al.), 5,207,915 (Hagen et al.), and 5,238,621 (Hagen et al.). Such materials are commercially available from 3M
Company 25 (St. Paul, MN) under the trade designations SDB-RPS (Styrene-Divinyl Benzene Reverse Phase Sulfonate, 3M Part No. 2241), cation-SR membrane (3M Part No. 2251), C-8 membrane (3M Part No. 2214), and anion-SR membrane (3M Part No. 2252).
Those that include a polytetrafluoroethylene matrix (PTFE) are particularly preferred. For example, U.S. Pat. No. 4,810,381 (Hagen et al.) discloses a solid phase 3o material that includes: a polytetrafluoroethylene fibril matrix, and nonswellable sorptive particles enmeshed in the matrix, wherein the ratio of nonswellable sorptive particles to polytetrafluoroethylene being in the range of 19:1 to 4:1 by weight, and further wherein the composite solid phase material has a net surface energy in the range of 20 to milliNewtons per meter. U.S. Pat. No. RE 36,811 (Markell et al.) discloses a solid phase extraction medium that includes: a PTFE fibril matrix, and sorptive particles enmeshed in the matrix, wherein the particles include more than 30 and up to 100 weight percent of porous organic particles, and less than 70 to 0 weight percent of porous (organic-coated or uncoated) inorganic particles, the ratio of sorptive particles to PTFE being in the range of 40:1 to 1:4 by weight.
Particularly preferred solid phase materials are available under the trade to designation EMPORE from the 3M Company, St. Paul, MN. The fundamental basis of the EMPORE technology is the ability to create a particle-loaded membrane, or disk, using any sorbent particle. The particles are tightly held together within an inert matrix of polytetrafluoroethylene (90% sorbent: 10% PTFE, by weight). The PTFE fibrils do not interfere with the activity of the particles in any way. The EMPORE membrane i5 fabrication process results in a denser, more uniform extraction medium than can be achieved in a traditional Solid Phase Extraction (SPE) column or cartridge prepared with the same size particles.
In another preferred embodiment, the solid phase (e.g., a microporous thermoplastic polymeric support) has a microporous structure characterized by a 2o multiplicity of spaced, randomly dispersed, nonuniform shaped, equiaxed particles of thermoplastic polymer connected by fibrils. Particles are spaced from one another to provide a network of micropores therebetween. Particles are connected to each other by fibrils, which radiate from each particle to the adjacent particles. Either, or both, the particles or fibrils may be hydrophobic. Examples of preferred such materials have a high 25 surface area, often as high as 40 meters2/gram as measured by Hg surface area techniques and pore sizes up to about S microns.
This type of fibrous material can be made by a preferred technique that involves the use of induced phase separation. This involves melt blending a thermoplastic polymer with an immiscible liquid at a temperature sufficient to form a homogeneous mixture, 30 forming an article from the solution into the desired shape, cooling the shaped article so as to induce phase separation of the liquid and the polymer, and to ultimately solidify the polymer and remove a substantial portion of the liquid leaving a microporous polymer matrix. This method and the preferred materials are described in detail in U.S. Patent Nos.
4,726,989 (Mrozinski), 4,957,943 (McAllister et al.), and 4,539,256 (Shipman).
Such materials are referred to as thermally induced phase separation membranes (TIPS
membranes) and are particularly preferred.
Other suitable solid phase materials include nonwoven materials as disclosed in U.S. Pat. No. 5,328,758 (Markell et al.). This material includes a compressed or fused particulate-containing nonwoven web (preferably blown microfibrous) that includes high sorptive-efficiency chromatographic grade particles.
Other suitable solid phase materials include those known as HIDE Foams, which are described, for example, in U.S. Pat. Publication No. 2003/0011092 (Tan et al.).
"HIDE" or "high internal phase emulsion" means an emulsion that includes a continuous reactive phase, typically an oil phase, and a discontinuous or co-continuous phase immiscible with the oil phase, typically a water phase, wherein the immiscible phase includes at least 74 volume percent of the emulsion. Many polymeric foams made from HIPE's are typically relatively open-celled. This means that most or all of the cells are in unobstructed communication with adjoining cells. The cells in such substantially open-celled foam structures have intercellular windows that are typically large enough to permit fluid transfer from one cell to another within the foam structure.
2o The solid phase material can include capture sites for inhibitors. Herein, "capture sites" refer to groups that are either covalently attached (e.g., functional groups) or molecules that are noncovalently (e.g., hydrophobically) attached to the solid phase material.
Preferably, the solid phase material includes functional groups that capture the inhibitors. For example, the solid phase material may include chelating groups. In this context, "chelating groups" are those that are polydentate and capable of forming a chelation complex with a metal atom or ion (although the inhibitors may or may not be .
retained on the solid phase material through a chelation mechanism). The incorporation of chelating groups can be accomplished through a variety of techniques. For example, a nonwoven material can hold beads functionalized with chelating groups.
Alternatively, the fibers of the nonwoven material can be directly functionalized with chelating groups.
Examples of chelating groups include, for example, -(CHZ-C(O)OH)2 , tris(2-aminoethyl)amine groups, iminodiacetic acid groups, nitrilotriacetic acid groups. The chelating groups can be incorporated into a solid phase material through a variety of techniques. They can be incorporated in by chemically synthesizing the material.
Alternatively, a polymer containing the desired chelating groups can be coated (e.g:, pattern coated) on an inert substrate (e.g., polycarbonate or polyethylene).
If desired, the substrate can be microreplicated for increased surface area and enhanced clean-up. It can also be pretreated with oxygen plasma, e-beam or ultraviolet radiation, heat, or a corona treatment process. This substrate can be used, for example, as a cover film, or laminated 1o to a cover film, on a reservoir in a microfluidic device.
Chelating solid phase materials are commercially available and could be used as the solid phase material in the present invention. For example, for certain embodiments of the present invention, EMPORE membranes that include chelating groups such as iminodiacetic acid (in the form of the sodium salt) are preferred. Examples of such membranes are disclosed in U.S. Pat. No. 5,147,539 (Hagen et al.) and commercially available as EMPORE Extraction Disks (47 mm, No. 2271 or 90 mm, No. 2371) from the 3M Company. For certain embodiments of the present invention, ammonium-derivatized EMPORE membranes that include chelating groups are preferred. To put the disk in the ammonium form, it can be washed with 50 mL of O.1M ammonium acetate buffer at pH
5.3 followed with several reagent water washes.
Examples of other chelating materials include, but are not limited to, crosslinked polystyrene beads available under the trade designation CHELEX from Bio-Rad Laboratories, Inc. (Hercules, CA), crosslinked agarose beads with tris(2-aminoethyl)amine, iminodiacetic acid, nitrilotriacetic acid,polyamines and polyimines as well as the chelating ion exchange resins commercially available under the trade designation DUOLITE
and DUOLITE GT73 from Rohm and Haas (Philadelphia, PA), AMBERLITE IRC-748, DIAION CR11, DUOLITE C647.
CHELEX 100 chelating resin, a styrene divinyl benzene copolymer containing iminodiacetate groups (-N-(CHZ-C(O)OH)2), has a high affinity for polyvalent metal ions 3o and can be used in certain methods of the present invention, although it is less desirable in methods carned out in microfluidic devices.
Typically, a desired concentration density of chelating groups on the solid phase material is about 0.02 nanomole per millimeter squared, although it is believed that a wider range of concentration densities is possible.
Other types of capture materials include anion exchange materials, cation exchange materials, activated carbon, reverse phase, normal phase, styrene-divinyl benzene, alumina, silica, zirconia, metal colloids. Examples of suitable anion exchange materials include strong anion exchangers such as quaternary ammonium, dimethylethanolamine, quaternary alkylamine, trimethylbenzyl ammonium, and dimethylethanolbenzyl ammonium usually in the chloride form, and weak anion exchangers such as polyamine.
Examples of suitable cation exchange materials include strong cation exchangers such as sulfonic acid typically in the sodium form, and weak cation exchangers such as carboxylic acid typically in the hydrogen form. Examples of suitable carbon-based materials include EMPORE carbon materials, carbon beads, Examples of suitable reverse phase C8 and C18 materials include silica beads that are end-capped with octadecyl groups or octyl is groups and EMPORE materials that have C8 and C18 silica beads (EMPORE
materials are available from 3M Co., St. Paul, MN). Examples of normal phase materials include hydroxy groups and dihydroxy groups.
Commercially available materials can also be modified or directly used in methods of the present invention, particularly in microfluidic devices. For example, solid phase 2o materials available under the trade designation LYSE AND GO (Pierce, Rockford, IL), RELEASE-IT (CPG, NJ), GENE FIZZ (Eurobio, France), GENE RELEASER
(Bioventures Inc., Murfreesboro, TN), and BUGS N BEADS (GenPoint, Oslo, Norway), as well as Zymo's beads (Zymo Research, Orange, CA) and Dynal's beads (Dynal, Oslo, Norway) can be incorporated into the methods of the present invention, particularly into a 25 microfluidic device as the solid phase capture material.
In certain embodiments of such methods, the solid phase material includes a coating reagent. The coating reagent is preferably selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric burner, and combinations thereof. In certain embodiments of such methods, the solid phase material 30 includes a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material, wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric burner, and combinations thereof.
Examples of suitable surfactants are listed below.
Examples of suitable strong bases include NaOH, KOH, LiOH, NH40H, as well as primary, secondary, or tertiary amines.
Examples of suitable polyelectrolytes include, polystryene sulfonic acid (e.g., poly(sodium 4-styrenesulfonate) or PSSA), polyvinyl phosphonic acid, polyvinyl boric acid, polyvinyl sulfonic acid, polyvinyl sulfuric acid, polystyrene phosphonic acid, polyacrylic acid, polymethacrylic acid, lignosulfonate, carrageenan, heparin, chondritin l0 sulfate, and salts or other derivatives thereof.
Examples of suitable selectively permeable polymeric barriers include polymers such as acrylates, acryl amides, azlactones, polyvinyl alcohol, polyethylene imine, polysaccharides. Such polymers can be in a variety of forms. They can be water-soluble, water-swellable, water-insoluble, hydrogels, etc. For example, a polymeric barrier can be prepared such that it acts as a filter for larger particles such as white blood cells, nuclei, viruses, bacteria, as well as nucleic acids such as human genomic DNA and proteins These surfaces could be tailored by one of skill in the art to separate on the basis of size and/or charge by appropriate selection of functional groups, by cross-linking, and the like.
Such materials would be readily available or prepared by one of skill in the art.
2o Preferably, the solid phase material is coated with a surfactant without washing any surfactant excess away, although the other coating reagents can be rinsed away if desired.
Typically, the coating can be carried out using a variety of methods such as dipping, rolling, spraying, etc. The coating reagent-loaded solid phase material is then typically dried, for example, in air, prior to use.
Particularly desirable are solid phase materials that are coated with a surfactant, preferably a nonionic surfactant. This can be accomplished according to the procedure set forth in the Examples Section. Although not intending to be limited by theory, the addition of the surfactant is believed to increase the wettability of the solid phase material, which allows the inhibitors to soak into the solid phase material and bind thereto.
3o The coating reagent for the solid phase materials are preferably aqueous-based solutions, although organic solvents (alcohols, etc.) can be used, if desired.
The coating reagent loading should be sufficiently high such that the sample is able to wet out the solid phase material. It should not be so high, however, that there is significant elution of the coating reagent itself. Preferably, if the coating reagent is eluted with the nucleic acid, there is no more than about 2 wt-% coating reagent in the eluted sample.
Typically, the coating solution concentrations can be as low as 0.1 wt-% coating reagent in the solution and as high as 10 wt-% coating reagent in the solution.
SURFACTANTS
Nonionic Surfactants. A wide variety of suitable nonionic surfactants are known that can be used as a lysing reagent (discussed above), an eluting reagent (discussed below), and/or as a coating on the solid phase material. They include, for example, polyoxyethylene surfactants, carboxylic ester surfactants, carboxylic amide surfactants, etc.
Commercially available nonionic surfactants include, n-dodecanoylsucrose, n-dodecyl-(3-D-glucopyranoside, n-octyl-(3-D-maltopyranoside, n-octyl-~3-D-thioglucopyranoside, n-decanoylsucrose, n-decyl-(3-D-maltopyranoside, n-decyl-(3-D-thiomaltoside, n-heptyl-~3-D-glucopyranoside, n-heptyl-~3-D-thioglucopyranoside, n-hexyl-~3-D-glucopyranoside, n-nonyl-~3-D-glucopyranoside, n-octanoylsucrose, n-octyl-~3-D-glucopyranoside, cyclohexyl-n-hexyl-(3-D-maltoside, cyclohexyl-n-methyl-(3-D-maltoside, digitonin, and those available under the trade designations PLURONIC, TRTTON, TWEEN, as well as numerous others commercially available and listed in the Kirk Othmer Technical Encyclopedia.
Examples are listed in Table 1 below. Preferred surfactants are the polyoxyethylene surfactants.
More preferred surfactants include octyl phenoxy polyethoxyethanol.
Table 1 SURFACTANT
TRADE NAME NONIONIC SURFACTANT SUPPLIER
PLURONIC F127 Modified oxyethylated alcohol Sigma and/or ox ro lated strai ht chain St. Louis, alcohols MO
TWEEN 20 Polyoxyethylene (20) sorbitan Sigma monolaurate St. Louis, MO
TRITON X-100 t-Octyl phenoxy polyethoxyethanolSigma St. Louis, MO
BRIJ 97 ~ Polyoxyethylene (10) oleyl Sigma ether ~
St. Louis, MO
IGEPAL CA-630 Octyl phenoxy poly (ethyleneoxy)Sigma ethanol St. Louis, MO
TOMADOL 1-7 Ethoxylated alcohol Tomah Products Milton, WI
Vitamin E TPGSd-Alpha tocopheryl polyethyleneEastman glycol 1000 Kingsport, TN
Also suitable are fluorinated nonionic surfactants of the type disclosed in U.S. Pat.
Publication Nos. 2003/0139550 (Savu et al.) and 2003/0139549 (Savu et al.).
Other nonionic fluorinated surfactants include those available under the trade designation ZONYL from DuPont (Wilmington, DE).
Zwitterionic Surfactants. A wide variety of suitable zwitterionic surfactants are known that can be used as a coating on the solid phase material, as a lysing reagent, and/or as an eluting reagent. They include, for example, alkylamido betaines and amine oxides thereof, alkyl betaines and amine oxides thereof, sulfo betaines, hydroxy sulfo betaines, amphoglycinates, amphopropionates, balanced amphopolycarboxyglycinates, and alkyl polyaminoglycinates. Proteins have the ability of being charged or uncharged depending on the pH; thus, at the right pH, a protein, preferably with a pI of about 8 to 9, such as modified Bovine Serum Albumin or chymotrypsinogen, could function as a zwitterionic surfactant. A specific example of a zwitterionic surfactant is cholamido propyl dimethyl ammonium propanesulfonate available under the trade designation CHAPS from Sigma.
More preferred surfactants include N-dodecyl-N,N dimethyl- 3- ammonia-1-propane sulfonate.
Cationic Surfactants. A wide variety of suitable cationic surfactants are known that can be used as a lysing reagent, an eluting reagent, andlor as a coating on the solid phase material. They include, for example, quaternary ammonium salts, polyoxyethylene alkylamines, and alkylamine oxides. Typically, suitable quaternary ammonium salts include at least one higher molecular weight group and two or three lower molecular weight groups are linked to a common nitrogen atom to produce a cation, and wherein the electrically-balancing anion is selected from the group consisting of a halide (bromide, chloride, etc.), acetate, nitrite, and lower alkosulfate (methosulfate, etc.).
The higher molecular weight substituent(s) on the nitrogen is/are often (a) higher alkyl group(s), containing about 10 to about 20 carbon atoms, and the lower molecular weight substituents may be lower alkyl of about 1 to about 4 carbon atoms, such as methyl or ethyl, which may be substituted, as with hydroxy, in some instances. One or more of the substituents may include an aryl moiety or may be replaced by an aryl, such as benzyl or phenyl. Among the possible lower molecular weight substituents are also lower alkyls of about 1 to about 4 carbon atoms, such as methyl and ethyl, substituted by lower polyalkoxy moieties such as polyoxyethylene moieties, bearing a hydroxyl end group, and falling within the general formula:
R(CHZCH20)~"_I~CH2CH 20H
where R is a (C1-C4)divalent alkyl group bonded to the nitrogen, and n represents an integer of about 1 to about 15. Alternatively, one or two of such lower polyalkoxy moieties having terminal hydroxyls may be directly bonded to the quaternary nitrogen instead of being bonded to it through the previously mentioned lower alkyl.
Examples of useful quaternary ammonium halide surfactants for use in the present invention include but are not limited to methyl- bis(2-hydroxyethyl)coco-ammonium chloride or oleyl-ammonium chloride, (ETHOQUAD C/12 and O/12, respectively) and methyl polyoxyethylene (15) octadecyl ammonium chloride (ETHOQUAD 18/25) from Akzo 2o ChemicalInc.
Anionic Surfactants. A wide variety of suitable anionic surfactants are known that can be used as a lysing reagent, an eluting reagent, and/or as a coating on the solid phase material. Surfactants of the anionic type that are useful include sulfonates and sulfates, such as alkyl sulfates, alkylether sulfates, alkyl sulfonates, alkylether sulfonates, alkylbenzene sufonates, alkylbenzene ether sulfates, alkylsulfoacetates, secondary alkane sulfonates, secondary alkylsulfates and the like. Many of these can include polyalkoxylate groups (e.g., ethylene oxide groups and/or propylene oxide groups, which can be in a random, sequential, or block arrangement) and/or cationic counterions such as Na, K, Li, 3o ammonium, a protonated tertiary amine such as triethanolamine or a quaternary ammonium group. Examples include: alkyl ether sulfonates such as lauryl ether sulfates available under the trade designation POLYSTEP B 12 and B22 from Stepan Company, Northfield, IL, and sodium methyl taurate available under the trade designation NIKKOL
CMT30 from Nikko Chemicals Co., Tokyo, Japan); secondary alkane sulfonates available under the trade designation HOSTAPUR SAS, which is a sodium (C 14-C
17)secondary alkane sulfonates (alpha-olefin sulfonates), from Clariant Corp., Charlotte, NC; methyl-2-sulfoalkyl esters such as sodium methyl-2-sulfo(C 12-C 16)ester and disodium 2-sulfo(C 12-C16)fatty acid available from Stepan Company under the trade designation ALPHASTE
PC-48; alkylsulfoacetates and alkylsulfosuccinates available as sodium laurylsulfoacetate (trade designation LANTHANOL LAL) and disodiumlaurethsulfosuccinate (trade 1o designation STEPANMILD SL3), both from Stepan Co.; and alkylsulfates such as ammoniumlauryl sulfate commercially available under the trade designation STEPANOL
AM from Stepan Co.
Another class of useful anionic surfactants include phosphates such as alkyl phosphates, alkylether phosphates, aralkylphosphates, and aralkylether phosphates. Many of these can include polyalkoxylate groups (e.g., ethylene oxide groups and/or propylene oxide groups, which can be in a random, sequential, or block arrangement).
Examples include a mixture of mono-, di- and tri-(alkyltetraglycolether)-o-phosphoric acid esters generally referred to as trilaureth-4-phosphate commercially available under the trade designation HOSTAPHAT 340KL from Clariant Corp., and PPG-5 ceteth 10 phosphate 2o available under the trade designation CRODAPHOS SG from Croda Inc., Parsipanny, NJ, as well as alkyl and alkylamidoalkyldialkylamine oxides. Examples of amine oxide surfactants include those commercially available under the trade designations AMMONYX LO, LMDO, and CO, which are lauryldimethylamine oxide, laurylamidopropyldimethylamine oxide, and cetyl amine oxide, all from Stepan Co.
ELUTION TECHNIQUES
For embodiments that use a solid phase material for retaining inhibitors, the more concentrated region of the sample that includes nucleic acid-containing material (e.g., nuclei) and/or released nucleic acid can be eluted using a variety of eluting reagents. Such 3o eluting reagents can include water (preferably RNAse-free sterile water), a buffer, a surfactant, which can be cationic, anionic, nonionic, or zwitterionic, or a strong base.
Preferably the eluting reagent is basic (i.e., greater than 7). For certain embodiments, the pH of the eluting reagent is at least 8. For certain embodiments, the pH
of the eluting reagent is up to 10. For certain embodiments, the pH of the eluting reagent is up to 13. If the eluted nucleic acid is used directly in an amplification process such as PCR, the eluting reagent should be formulated so that the concentration of the ingredients will not inhibit the enzymes (e.g., Taq Polymerise) or otherwise prevent the amplification reaction.
Examples of suitable surfactants include those listed above, particularly, those known as SDS, TRITON X-100, TWEEN, fluorinated surfactants, and PLURONICS. The l0 surfactants are typically provided in aqueous-based solutions, although organic solvents (alcohols, etc.) can be used, if desired. The concentration of a surfactant in an eluting reagent is preferably at least 0.1 weight/volume percent (w/v-%), based on the total weight of the eluting reagent. The concentration of a surfactant in an eluting reagent is preferably no greater than 1 w/v-%, based on the total weight of the eluting reagent. A
stabilizer, such as polyethylene glycol, can optionally be used with a surfactant.
Examples of suitable elution buffers include TRIS-HCI, N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), 3-[N-morpholino]propanesulfonic acid (MOPS), piperazine-N,N'-bis[2-ethanesulfonic acid]
(PIPES), 2-[N-morpholino]ethansulfonic acid (MES), TRIS-EDTA (TE) buffer, sodium citrate, ammonium acetate, carbonate salts, and bicarbonates etc.
The concentration of an elution buffer in an eluting reagent is preferably at least 10 millimolar (mM). The concentration of a surfactant in an eluting reagent is preferably no greater than 2 weight percent (wt-%).
Typically, elution of the nucleic acid-containing material and/or released nucleic acid is preferably accomplished using an alkaline solution. Although not intending to be bound by theory, it is believed that an alkaline solution allows for improved binding of inhibitors, as compared to elution with water. The alkaline solution also facilitates lysis of nucleic acid-containing material. Preferably, the alkaline solution has a pH
of 8 to 13, and more preferably 13. Examples of sources of high pH include aqueous solutions of NaOH, KOH, LiOH, quaternary nitrogen base hydroxide, tertiary, secondary or primary amines, etc. If an alkaline solution is used for elution, it is typically neutralized in a subsequent step, for example, with TRIS buffer, to form a PCR-ready sample.
The use of an alkaline solution can selectively destroy RNA, to allow for the analysis of DNA. Otherwise, RNAse can be added to the formulation to inactivate RNA, followed by heat inactivation of the RNAse. Similarly, DNAse can be added to selectively destroy DNA and allow for the analysis of RNA; however, other lysis buffers (e.g., TE) that do not destroy RNA would be used in such methods. The addition of RNAse inhibitor such as RNAsin can also be used in a formulation for an RNA preparation that is subjected to real-time PCR.
to Elution is typically carned out at room temperature, although higher temperatures may produce higher yields. For example, the temperature of the eluting reagent can be up to 95°C if desired. Elution is typically carried out within 10 minutes, although 1-3 minute elution times are preferred.
Examples of devices for using the methods of the present invention include standard laboratory filter holders furnished by companies such as Millipore, Inc. (Bedford, MA), Bio-Rad, Inc. (Hercules, CA), Osmonics, Inc. (Westborough, MA), and Whatman, Inc. (Clifton, NJ). The method of the invention can be conducted in filtration devices 2o which facilitate the movement of solutions through solid phase materials (referred to as flow-through devices) by means including centrifugation, suction, pressure.
Other devices include microtiter plates and microfluidic devices.
The present invention also provides a kit, which can include a solid phase material either with or without a holder (for example, a filter holder such as a syringe filter holder 25 or a spin filter holder, or a column with retaining frits at each end for retaining particulate material), a lysing reagent (particularly a surfactant such as a nonionic surfactant, either neat or in a solution), and instructions for binding inhibitors and eluting the nucleic acid.
Preferably, the present invention provides kits that include a flow-through receptacle (more preferably, a microfluidic device) having a solid phase (preferably, polymeric) 3o material therein, and preferably a nonionic surfactant.
Other components that could be included within kits of the present invention include conventional reagents such as wash solutions, coupling buffers, quenching buffers, blocking buffers, elution buffers, and the like. Other components that could be included within kits of the present invention include conventional equipment such as spin columns, cartridges, 96-well filter plates, syringe filters, collection units, syringes, and the like.
The kits typically include packaging material, which refers to one or more physical structures used to house the contents of the kit. The packaging material can be constructed by well-known methods, preferably to provide a sterile, contaminant-free environment.
The packaging material may have a label that indicates the contents of the kit. 1n addition, the kit contains instructions indicating how the materials within the kit are employed. As used herein, the term "package" refers to a solid matrix or material such as glass, plastic, paper, foil, and the like.
"Instructions" typically include a tangible expression describing the various methods of the present invention, including lysing conditions (e.g., lysing reagent type and concentration), the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like.
Although the methods can be used in a variety of devices, a variety of illustrative embodiments of preferred devices are described in U.S. Patent Publication Nos.
2002/0047003 (published April 25, 2003, Bedingham et al.).
2o Preferred devices useful in methods of the present invention include microfluidic devices. These typically employ a body structure that has an integrated microfluidic channel network disposed therein. In preferred aspects, the body structure of the microfluidic devices include an aggregation of two or more separate layers which, when appropriately mated or joined together, form the microfluidic device of the invention, e.g., containing the channels and/or chambers described herein. Typically, useful microfluidic devices include a top portion, a bottom portion, and an interior portion, wherein the interior portion substantially defines the channels and chambers of the device. Typically, the chambers include valves (e.g., valve septums) and are referred to as valued chambers.
A particularly preferred device for certain embodiments herein is referred to as a 3o variable valve device and is disclosed in Applicants' Assignee's copending U.S. Patent Application Serial No. 10/734,717, filed on December 12, 2003, entitled Variable Valve Apparatus and Method. In this variable valve device, the valve structures allow for removal of selected portions of the sample material located within the process chamber (i.e., the variable valued process chamber). Removal of the selected portions is achieved by forming an opening in a valve septum at a desired location.
The valve septums are preferably large enough to allow for adjustment of the location of the opening based on the characteristics of the sample material in the process chamber. If the sample processing device is rotated after the opening is formed, the selected portion of the material located closer to the axis of rotation exits the process chamber through the opening. The remainder of the sample material cannot exit through to the opening because it is located farther from the axis of rotation than the opening.
The openings in the valve septum may be formed at locations based on one or more characteristics of the sample material detected within the process chamber. It may be preferred that the process chambers include detection windows that transmit light into and/or out of the process chamber. Detected characteristics of the sample material may 15 include, e.g., the free surface of the sample material (indicative of the volume of sample material in the process chamber). Forming an opening in the valve septum at a selected distance radially outward of the free surface can provide the ability to remove a selected volume of the sample material from the process chamber.
For sample materials that can be separated into various components, e.g., whole 2o blood, rotation of the sample processing device may result in separation of the plasma and red blood cell components, thus allowing for selective removal of the components to, e.g., different process chambers.
In some embodiments, it may be possible to remove selected aliquots of the sample material by forming openings at selected locations in one or more valve septums. The 25 selected aliquot volume can be determined based on the radial distance between the openings (measured relative to the axis of rotation) and the cross-sectional area of the process chamber between the opening.
The openings in the valve septums are preferably formed in the absence of physical contact, e.g., through laser ablation, focused optical heating, etc. As a result, the openings 3o can preferably be formed without piercing the outermost layers of the sample processing device, thus limiting the possibility of leakage of the sample material from the sample processing device.
In one aspect, the present invention uses a valued process chamber in a sample processing device (e.g., a microfluidic device), the valued process (e.g., heating, mixing, lysing, combining fluids) chamber including a process chamber having a process chamber volume located between opposing first and second major sides.of the sample processing device, wherein the process chamber occupies a process chamber area in the sample processing device, and wherein the process chamber area has a length and a width transverse to the length, and further wherein the length is greater than the width. The variable valued process chamber also includes a valve chamber located within the process chamber area, the valve chamber located between the process chamber volume and the second major side of the sample processing device, wherein the valve chamber is isolated from the process chamber by a valve septum separating the valve chamber and the process chamber, and wherein a portion of the process chamber volume lies between the valve septum and a first major side of the sample processing device. A detection window is located within the process chamber area, wherein the detection window is transmissive to selected electromagnetic energy directed into and/or out of the process chamber volume.
In another aspect, the present invention provides a method that allows for the selective removal of a portion of a sample from a variable valued process chamber. The 2o method includes providing a sample processing device (e.g., a microfluidic device) as described above, providing sample material in the process chamber; detecting a characteristic of the sample material in the process chamber through the detection window;
and forming an opening in the valve septum at a selected location along the length of the process chamber, wherein the selected location is correlated to the detected characteristic of the sample material. The method also includes moving only a portion of the sample material from the process chamber into the valve chamber through the opening formed in the valve septum 3o In one embodiment, the present invention provides a method of isolating nucleic acid from a sample. In this illustrative method, the inhibitors are contained within cells, but it is understood that this may not always be the case, and methods of the present invention can be used with such samples.
The method includes: providing a sample including nucleic acid-containing material and cells containing inhibitors (such nucleic acid-containing material and cells containing inhibitors may be the same or different); contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample including nucleic acid-containing material and inhibitors; forming a concentrated region of the lysed sample; wherein the concentrated region of the lysed sample includes nucleic acid-containing material and inhibitors; substantially separating 1 o the concentrated region from a less concentrated region of the lysed sample; contacting the separated concentrated region of the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material includes a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barner, and combinations thereof;
optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nucleic acid-containing 2o material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
The nucleic acid-containing material and cells containing inhibitors may be the same or different, although they are typically different. That is, the nucleic acid containing material and the inhibitor-containing cells could potentially be the same. For example, if the sample is a huffy coat, the nucleic acid containing material can be a white blood cell, which includes both nuclei and inhibitors. If a lysing reagent (e.g., a nonionic surfactant) is used that will lyse the cell membranes of the white blood cells but not the nuclei included therein, then the inhibitors are released as are in-tact nuclei, which is also considered to be nucleic acid-containing material as defined herein.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method includes: providing a sample including cells containing inhibitors and cells containing nuclei (such cells containing inhibitors and cells containing nuclei may be the same or different); contacting the biological sample with a nonionic surfactant under conditions effective to break cell membranes and release nuclei and inhibitors and form a lysed sample; forming a concentrated region of the lysed sample;
wherein the concentrated region of the sample includes nuclei and inhibitors;
substantially separating the concentrated region of the lysed sample from the less concentrated region of the sample; contacting the separated concentrated region of the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material includes a polytetrafluoroethylene fibril matrix, to sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric burner, and combinations thereof; optionally further lysing the nuclei to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nucleic acid-containing and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
The cells containing inhibitors and cells containing nuclei may be the same or different, although they are typically different cells. That is, the nuclei-containing cells 2o and the inhibitor-containing cells could potentially be the same. For example, the inhibitors could include nuclear proteins, as well as cellular proteins.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method includes: providing a microfluidic device including a loading chamber, a valued process chamber, and a separation chamber including a solid phase material; providing a sample including nucleic acid-containing material and cells containing inhibitors (such nucleic acid-containing material and cells containing inhibitors may be the same or different); placing the sample in the loading chamber; contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample including nucleic 3o acid-containing material and inhibitors; transferring the lysed sample to a valued process chamber; forming a concentrated region of the lysed sample in the valued process chamber, wherein the concentrated region of the lysed sample includes nucleic acid-containing material and inhibitors; substantially separating the concentrated region from a less concentrated region of the lysed sample; transferring the separated concentrated region of the lysed sample to the separation chamber for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material includes capture sites (preferably chelating functional groups), a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof; optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto. As discussed above, the nucleic acid-containing material and cells containing inhibitors may be the same or different, although they are typically different.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method includes: providing a microfluidic device including a loading chamber, a valued process chamber, and a separation chamber including a solid phase material; providing a sample including cells containing inhibitors and cells containing nuclei (such cells containing inhibitors and cells containing nuclei may be the same or different); placing the sample in the loading chamber;
contacting the sample with a nonionic surfactant under conditions effective to break cell membranes and release nuclei and inhibitors to form a lysed sample; transferring the lysed sample to a valued process chamber; forming a concentrated region of the lysed sample in the valued process chamber, wherein the concentrated region of the lysed sample includes nuclei and inhibitors; substantially separating the concentrated region from a less concentrated region of the lysed sample; transferring the separated concentrated region of the lysed sample to the separation chamber for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material includes capture sites (preferably chelating functional groups), a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof; optionally further lysing the nuclei to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nucleic acid-containing and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto. As discussed above, the cells containing inhibitors and cells containing nuclei may be the same or different, although they are typically different cells.
In certain embodiments of such methods, the nucleic acid-containing material l0 includes nuclei and the method includes separating at least a portion of the nuclei from the solid phase material. That is, in-tact nuclei contact the solid phase material.
In certain embodiments of such methods, separating at least a portion of the nucleic acid-containing material (e.g., nuclei) and/or nucleic acid from the solid phase material includes transferring the nucleic acid-containing material (e.g., nuclei) and/or nucleic acid to an amplification reaction chamber (e.g., PCR sample chamber).
In certain embodiments of such methods, the coating reagent on the solid phase material includes a surfactant. Preferably, the surfactant is a nonionic or a zwitterionic surfactant, and more preferably, a nonionic surfactant.
In certain embodiments of such methods, separating at least a portion of the nucleic acid-containing material (e.g., nuclei) and/or nucleic acid includes eluting at least a portion of the nucleic acid-containing material (e.g., nuclei) and/or nucleic acid from the solid phase material with an eluting reagent.
In certain embodiments of such methods of the invention, if desired the methods can include further lysing the nucleic acid-containing material (e.g., nuclei) using a second lysing reagent.
Alternatively, in certain embodiments of such methods, the eluting reagent is also a lysing reagent. For elution and lysing simultaneously, a strong base, such as NaOH is preferred. For elution without lysing, water or TE buffer is preferred. For elution of a prelysed sample, any elution reagent (i.e., eluting reagent) can be used.
Refernng to Figure 1, a preferred embodiment of the microfluidic device suitable for use with these embodiments includes a loading chamber 10, an optional mixing chamber 12, a valued process chamber 14, a separation chamber 16 that includes a solid phase material, an eluting reagent chamber 18, a waste chamber 20 and an optional amplification reaction chamber 22. A microfluidic device that includes such a valued process chamber is disclosed, for example, in Applicants' Assignee's copending U.S.
Patent Application Serial No. 10/734,717, filed on December 12, 2003, entitled Variable Valve Apparatus and Method. These chambers are in fluid communication with each other such that a sample can be loaded into the loading chamber 10, which can then be transferred to the mixing chamber 12, or if it is not present, directly to the valued process chamber 14. If the sample is premixed with a lysing reagent (i.e., pre-lysed), for example, to then the mixing chamber 12 may not be needed. Alternatively, the mixing chamber 12 could include a lysing reagent, for example, in a pre-deposited (and typically, a dried-down) form. The sample can be concentrated in the valued process chamber 14 by a variety of means, typically by centrifugation. The valve of the valued process chamber 14 is typically positioned such that a concentrated region of a sample (that includes target nucleic acid-containing material and/or nucleic acid and inhibitors) can be substantially separated from the less concentrated region of the sample (that can also include inhibitors and a lesser amount of the target nucleic acid-containing material and/or nucleic acid).
The less concentrated region of the sample is typically transferred to the waste chamber 20. The more concentrated region of the sample is transferred to the separation chamber 16 for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material. The eluting reagent in the eluting reagent chamber 18 is then transferred to the separation chamber 16 to remove at least a portion of the target nucleic acid-containing material and/or nucleic acid. In some cases, it may be useful to archive the nuclei in the consumable for later use. In certain embodiments, this material can be directly transferred to an amplification reaction chamber 22 for carrying out a PCR
process, for example. The amplification reaction chamber 22 can optionally include pre-deposited (and typically, dried-down) reactants for the amplification reaction (e.g., PCR).
3o In another embodiment, the present invention provides a method of isolating nucleic acid from a sample. In this illustrative method, the inhibitors are contained within cells, but it is understood that this may not always be the case, and methods of the present invention can be used with such samples.
The method includes: providing a sample including nucleic acid-containing material and cells containing inhibitors (which may be the same or different);
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample including nucleic acid-containing material and inhibitors; contacting the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material includes a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the lysed sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic surfactant.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method includes: providing a sample including nucleic acid-containing material and cells containing inhibitors (which may be the same or different); contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample. including nucleic acid-containing material and inhibitors; contacting the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material includes a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barner, and 3o combinations thereof; separating at least a portion of the nucleic acid-containing material from the solid phase material having at least a portion of the inhibitors adhered thereto;
and after separating the nucleic acid-containing material, further lysing the nucleic acid-containing material to release nucleic acid.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method includes: providing a microfluidic device including a loading chamber and a separation chamber including a solid phase material;
providing a sample including nucleic acid-containing material and cells containing inhibitors (which may be the same or different); placing the sample in the loading chamber; contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample including nucleic acid-containing material and inhibitors; transferring the lysed sample to the separation chamber to contact the solid phase material and to preferentially adhere at least a portion of the inhibitors to the solid phase material; optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the sample with the solid phase material; wherein the solid phase material includes capture sites (e.g., chelating functional groups), a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barner, and combinations thereof; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic surfactant.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method includes: providing a microfluidic device including a loading chamber and a separation chamber including a solid phase material;
providing a sample including nucleic acid-containing material and cells containing inhibitors (which may be the same or different); placing the sample in the loading chamber; contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample including nucleic acid-containing material and inhibitors; transferring the lysed sample to the separation 3o chamber to contact the solid phase material and to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material includes capture sites (e.g., chelating functional groups), a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barner, and combinations thereof; and separating at least a portion of the nucleic acid-containing material from the solid phase material having at least a portion of the inhibitors adhered thereto; and after separating the nucleic acid-containing material, further lysing the nucleic acid-containing material to release nucleic acid.
As discussed above with respect to Illustrative Method l, the nucleic acid-containing material and cells containing inhibitors of the embodiments of Illustrative 1o Method 2 may be the same or different, although they are typically different. That is, the nucleic acid containing material and the inhibitor-containing cells could potentially be the same. Also, in illustrative Method 2, the sample can include free nucleic acid and free inhibitors.
In certain embodiments of such methods, separating the nucleic acid-containing material (e.g., nuclei) (and/or nucleic acid if already isolated from the nucleic acid-containing material) from the solid phase material includes transferring the nucleic acid-containing material (e.g., nuclei) (and/or nucleic acid) to an amplification reaction chamber (e.g., a PCR sample chamber).
In certain embodiments of such methods, the solid phase material includes a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material, wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof.
1n certain embodiments of such methods, separating includes eluting at least a portion of the nucleic acid-containing material (e.g., nuclei) (and/or nucleic acid) from the solid phase material with an eluting reagent.
Refernng to Figure 2, a preferred embodiment of the microfluidic device suitable for use with these embodiments includes a loading chamber 30, an optional mixing chamber 32, a separation chamber 36 that includes a solid phase material, an eluting reagent chamber 38, and an optional amplification chamber 42. These chambers are in fluid communication with each other such that a sample can be loaded into the loading chamber 30, which can then be transferred to the mixing chamber 32, or if it is not present, directly to the separation chamber 36. If the sample is premixed with a lysing reagent (i.e., pre-lysed), for example, then the mixing chamber 32 may not be needed.
Alternatively, the mixing chamber 32 could include a lysing reagent, for example, in a pre-deposited (and typically, a dried-down) form. The lysed sample is transferred to the separation chamber 36 for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material. The eluting reagent in the eluting reagent chamber 38 is then transferred to the separation chamber 36 to remove at least a portion of the target nucleic acid-containing material and/or nucleic acid. The sample can be eluted with io a strong base for further lysing. After lysis using a strong base, in some cases, the samples can be heated for a few minutes at high temperatures (95°C) to denature proteins that are inhibitory to PCR followed by neutralization using an agent such as TRIS
buffer to adjust the pH of the sample prior to PCR. Alternatively, the eluted sample can be transferred to a chamber for further lysing using other methods (e.g., heating, Proteinase K).
Alternatively, these reagents could be pre-deposited (and dried-down) if desired. In certain embodiments, this material can be directly transferred to an amplification reaction chamber 42 for carrying out a PCR process, for example. The amplification reaction chamber 42 can optionally include pre-deposited reactants for the amplification reaction (e.g., PCR).
ADDITIONAL EMBODIMENTS
In certain embodiments of such methods that involve the use of a microfluidic device, placing the sample in the loading chamber occurs prior to contacting the sample with a first lysing reagent. Alternatively, placing the sample in the loading chamber occurs after contacting the sample with a first lysing reagent. The first lysing reagent can be water or a nonionic surfactant, for example.
If additional lysing is needed to release nucleic acid from nucleic acid-containing material (e.g., nuclei), other lysing conditions can be used. For example, this includes subjecting the nucleic acid-containing material to a strong base with optional heating. The 3o strong base is typically NaOH, but can be others such as KOH, LiOH, NH40H, as well as primary, secondary, or tertiary amines. Typically, the temperature is at room temperature.
If a base is used, the sample containing the released nucleic acid may need to have its pH
adjusted, particularly if the nucleic acid is to be subjected to a subsequent amplification process. Thus, certain embodiments of the invention include adjusting the pH
of the sample typically to at least 7.5, and typically to no greater than 9.
In certain embodiments of such methods that involve the use of a microfluidic device, the first lysing reagent is a nonionic surfactant. In certain embodiments of such methods, the loading chamber includes the first lysing reagent (e.g., pre-deposited (and typically, dried-down) nonionic surfactant) and contacting the sample with a first lysing reagent occurs upon placing the sample in the loading chamber.
1o Alternatively, the sample could be transferred to a subsequent processing chamber with the first lysing reagent (preferably, a nonionic surfactant) therein. For example, contacting the sample with a first lysing reagent (preferably, a nonionic surfactant) occurs in a mixing chamber with sufficient mixing to break cell membranes and release nuclei and inhibitors to form a lysed sample. It should be understood that if a "first" lysing 15 reagent is used, the methods do not necessarily require the use of a "second" lysing reagent; rather, the term "first lysing reagent" is used herein to distinguish from any additional lysing reagents if used.
As stated above, the addition of sucrose in a buffer (particularly, a TRIS
buffer) may help in the isolation of nuclei. The buffer could also include magnesium salts and 2o surfactants such as TRITON X-100. This may also provide a good medium for lysis of white blood cells. Furthermore, in certain cases, when the nuclei need to be archived, particularly within a microfluidic device, using a nuclei storage buffer may be useful. The nuclei storage buffer could include sucrose, magnesium salts, EDTA, dithiothrietol, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), and/or glycerol, for example, in a buffer 25 (e.g., TRIS buffer) and would allow for stable storage of nuclei.
In certain embodiments of such methods that involve the use of a microfluidic device, forming a concentrated region of the sample in the valued process chamber includes centrifuging the sample in the process chamber. Typically, separating the concentrated region from the less concentrated of the sample includes transferring the less 3o concentrated region of the sample through the valve to a waste bin.
Spinning speeds, such as 400 rcf for 2 minutes, are typically desirable when using a microfluidic device, although higher spinning speeds andlor longer spinning times may be used.
The nonionic surfactant, such as TRITON X-100, can be pre-deposited (and dried down uniformly if desired) in a mixing chamber of a microfluidic device, for example, such that lysing occurs when the sample (e.g., blood) is mixed with the surfactant for a few minutes. In other cases, a dilute solution of surfactant can be pre-mixed with the sample (e.g., blood) prior to introduction into the mixing chamber.
In other cases, when a nonionic surfactant is not used, water or ammonium chloride can be used for lysis of red blood cells. After lysis occurs in the mixing chamber, the white blood cells can be centrifuged down at relatively low speeds.
In certain embodiments of the methods described herein, the sample can be whole blood. The whole blood is then typically separated into component parts and the portion containing white blood cells (typically referred to as the huffy coat) separated and lysed to release the nuclei and/or nucleic acid. For example, in certain embodiments, the method can include centrifuging the whole blood (e.g., in a valued process chamber) to form a plasma layer (often in the upper layer), a red blood cell layer (often in the lower layer), and an interfacial layer that includes white blood cells, and removing a substantial portion of the interfacial layer (i.e., huffy coat). The huffy coat can then be subjected to further processing.
In certain embodiments, the huffy coat could be separated from whole blood using conventional techniques. The huffy coat could then be used as the sample in the methods described herein.
In addition to the solid phase material discussed herein, other types of solid phase materials, particularly beads, can be introduced into a microfluidic device in a variety of embodiments of the present invention. For example, beads can be functionalized with the appropriate groups to isolate specific cells, viruses, bacteria, proteins, nucleic acids, etc.
The beads can be segregated from the sample by centrifugation and subsequent separation.
The beads could be designed to have the appropriate density and sizes (nanometers to microns) for segregation. For example, in the case of viral capture, beads that recognize the protein coat of a virus can be used to capture and concentrate the virus prior to or after removal of small amounts of residual inhibitors from a serum sample.
The inhibitors can be removed using solid phase materials, as described herein, prior to or after capture of viral particles onto the beads. In addition to this, the amount of inhibitors can be reduced using concentration/separation/optional dilution steps, for example, as disclosed in U.S. Patent Application Serial No. , filed on , entitled METHODS FOR NUCLEIC ACID ISOLATION AND KITS USING
A MICROFLUIDIC DEVICE AND CONCENTRATION STEP (Attorney Docket No.
59801US002). Such concentration/separation/optional dilution steps can be used with various methods and samples described herein.
Nucleic acids can be extracted out of the segregated viral particles by lysis.
Thus, the beads could provide a way of concentrating relevant material in a specific region within a microfluidic device, also allowing for washing of irrelevant materials and elution of relevant material from the captured particle.
Examples of such beads include, but are not limited to, crosslinked polystyrene beads available under the trade designation CHELEX from Bio-Rad Laboratories, Inc.
(Hercules, CA), crosslinked agarose beads with tris(2-aminoethyl)amine, iminodiacetic acid, nitrilotriacetic acid, polyamines and polyimines as well as the chelating ion exchange resins commercially available under the trade designation DUOLITE C-467 and DUOLITE GT73 from Rohm and Haas (Philadelphia, PA), AMBERLITE IRC-748, DIAION CR11, DUOLITE C647. These beads are also suitable for use as the solid phase 2o material as discussed above.
Other examples of beads include those available under the trade designations GENE FIZZ (Eurobio, France), GENE RELEASER (Bioventures Inc., Murfreesboro, TN), and BUGS N BEADS (GenPoint, Oslo, Norway), as well as Zymo's beads (Zymo Research, Orange, CA) and DYNAL beads (Dynal, Oslo, Norway).
Other materials are also available for pathogen capture (e.g., viral particles, bacteria). For example, polymer coatings can also be used to isolate specific cells, viruses, bacteria, proteins, nucleic acids, etc. in certain embodiments of the invention. These polymer coatings could directly be spray jetted, for example, onto the cover film of a microfluidic device.
3o Viral particles can be captured onto beads by covalently attaching antibodies onto bead surfaces. The antibodies can be raised against the viral coat proteins.
For example, DYNAL beads can be used to covalently link antibodies. Alternatively, synthetic polymers, for example, anion-exchange polymers, can be used to concentrate viral particles. Commercially available resins such as viraffinity (Biotech Support Group, East Brunswick, NJ) can be used to coat beads or applied as polymer coatings onto select locations in microfluidic device to concentrate viral particles. BUGS N BEADS
(GenPoint, Oslo, Norway) can, for example, be used for extraction of bacteria.
Here, these beads can be used to capture bacteria such as Staphylococcus, Streptococcus, E
coli, Salmonella, and Clamydia elementary bodies.
Thus, in one embodiment of the present invention when the sample includes viral l0 particles or other pathogens (e.g., bacteria), a microfluidic device can include solid phase material in the form of viral capture beads or other pathogen capture material. More specifically, in one case, the beads can be used only for concentration of virus or bacteria, for example, followed by segregation of beads to another chamber, ending with lysis of virus or bacteria. In another case, the beads can be used for concentration of virus or 15 bacteria, followed by lysis and capture of nucleic acids onto the same bead, dilution of beads, concentration of beads, segregation of beads, and repeating the process multiple times prior to elution of captured nucleic acid.
If the downstream application of the nucleic acid is subjecting it to an amplification process such as PCR, then all reagents used in the method are preferably compatible with 20 such process (e.g., PCR compatible). Furthermore, the addition of PCR
facilitators may be useful, especially for diagnostic purposes. Also, heating of the material to be amplified prior to amplification can be beneficial.
In embodiments in which the inhibitors are not completely removed, the use of buffers, enzymes, and PCR facilitators can be added that help in the amplification process 25 in the presence of inhibitors. For example, enzymes other than Taq Polymerase, such as rTth, that are more resistant to inhibitors can be used, thereby providing a huge benefit for PCR amplification. The addition of Bovine Serum Albumin, betaine, proteinase inhibitors, bovine transferrin, etc. can be used as they are known to help even further in the amplification process. Alternatively, one can use a commercially available product such as 30 Novagen's Blood Direct PCR Buffer kit (EMD Biosciences, Darmstadt, Germany) for direct amplification from whole blood without the need for extensive purification.
Objects and advantages of this invention are further illustrated by the following examples, but the particular materials and amounts thereof recited in these examples, as well as other conditions and details, should not be construed to unduly limit this invention.
EXAMPLES
Example lA: Preparation of Solid Phase Material: Ammonia Form without TRITON X-A 3M No. 2271 EMPORE Extraction Chelating Disk (3M, St. Paul, MN) was 1o placed in a glass filter holder. The extraction disk was converted into the ammonia form, following the procedure printed on the package insert.
Example 1B: Preparation of Solid Phase Material: Ammonia Form with TRITON X-A 3M No. 2271 EMPORE Extraction Chelating Disk was placed in a glass filter 15 holder. The extraction disk was converted into the ammonia form, following the procedure printed on the package insert. The disk placed in a vial and was submerged in a 1 TRITON X-100 (Sigma-Aldrich, St. Louis, MO) solution (0.1 gram (g) of TRITON X-in 10 mL of water), mixing for about 6-8 hours on a Thermolyne Vari-Mix Model Rocker (Barnstead/Thermolyne, Dubuque, IA). The disk was placed in glass filter holder, 2o dried by applying a vacuum for about 20 minutes (min), and then dried overnight at room temperature (approximately 21°C), taking care not to wash or rinse the disk.
Example 1 C: Preparation of Solid Phase Material: Anion-SR Empore Chelating Membrane with TRITON X-100 25 A 3M No. 2252 EMPORE Extraction Chelating Disk was placed in a vial and was submerged in a 1% TRITON X-100 solution (0.1 g of TRITON X-100 in 10 milliliters (mL) of water), mixing for about 6-8 hours on a Thermolyne Vari-Mix Model Rocker. The disk was placed in glass filter holder, dried by applying a vacuum for about 20 minutes, and then dried overnight at room temperature (approximately 21°C), taking 3o care not to wash or rinse the disk.
Example 1D: Preparation of Solid Phase Material: C8 Chelating Membrane with A 3M No. 2214 EMPORE Extraction Chelating Disk was placed in a vial and was submerged in a 1 % TRITON X-100 solution (0.1 g of TRITON X-100 in 10 mL of water), mixing for about 6-8 hours on a Thermolyne Vari-Mix Model M48725 Rocker. The disk was placed in glass filter holder, dried by applying a vacuum for about 20 minutes, and then dried overnight at room temperature (approximately 21°C), taking care not to wash or rinse the disk.
to Example lE: Preparation of Solid Phase Material: Chelating Membrane with TRITON X-100 and FR-2025 A 3M No. 2271 EMPORE Extraction Chelating Disk was placed in a vial and was submerged in a 50/50 mixture of 1% TRITON X-100 (0.1 g of TRITON X-100 in 10 15 milliliters (mL) of water) and 0.1% FR-2025 (3M, St. Paul, MN) and mixed for about 6-8 hours on a Thermolyne Vari-Mix Model M48725 Rocker. The disk was placed in glass filter holder, dried by applying a vacuum for about 20 minutes, and then dried overnight at room temperature (approximately 21°C), taking care not to wash or rinse the disk.
2o Example 1F: Preparation of Solid Phase Material: Chelating Membrane with TRITON X-100 and FC-4430 A 3M No. 2271 EMPORE Extraction Chelating Disk was placed in a vial and was submerged in a 50/50 mixture of 1% TRITON X-100 (0.1 g of TRITON X-100 in 10 milliliters (mL) of water) and 0.1 % FC-4430 (3M, St. Paul, MN) and mixed for about 6-8 25 hours on a Thermolyne Vari-Mix Model M48725 Rocker. The disk was placed in glass filter holder, dried by applying a vacuum for about 20 minutes, and then dried overnight at room temperature (approximately 21°C), taking care not to wash or rinse the disk.
Example 1 G: Preparation of Solid Phase Material: Chelating Membrane with TRITON
X- 100 and PSSA
A 3M No. 2271 EMPORE Extraction Chelating Disk was placed in a glass filter holder. The extraction disk was converted into the ammonia form, following the procedure printed on the package insert. The disk placed in a vial and was submerged in a 50/50 mixture of 1 % TRITON X-100 (0.1 g of TRITON X-100 in 10 milliliters (mL) of water) and 1% PSSA (Poly(sodium 4-styrene-sulfonate) (Sigma-Aldrich, St. Louis, MO) (20 wt-PSSA stock solution was diluted to 1 wt-% in RNAse-free sterile water) and mixed for about 6-8 hours on a Thermolyne Vari-Mix Model M48725 Rocker. The disk was placed l0 in glass filter holder, dried by applying a vacuum for about 20 minutes, and then dried overnight at room temperature (approximately 21°C), taking care not to wash or rinse the disk.
Example 1H: Preparation of Solid Phase Material: Cation-SR EMPORE Chelating Membrane with TRITON X-100 A 3M No. 2251 EMPORE Extraction Chelating Disk was placed in a vial and was submerged in a 1% TRITON X-100 solution (0.1 g of TRITON X-100 in 10 mL of water), mixing for about 6-8 hours on a Thermolyne Vari-Mix Model M48725 Rocker. The disk was placed in glass filter holder, dried by applying a vacuum for about 20 minutes, and then dried overnight at room temperature (approximately 21 °C), taking care not to wash or rinse the disk.
Example 1J: Preparation of Solid Phase Material: EMPORE Chelating Membrane with A 3M No. 2241 EMPORE Extraction Chelating Disk was placed in a vial and was submerged in a 1 % TRITON X-100 solution (0.1 g of TRITON X-100 in 10 mL of water), mixing for about 6-8 hours on a Thermolyne Vari-Mix Model M48725 Rocker. The disk was placed in glass filter holder, dried by applying a vacuum for about 20 minutes, and then dried overnight at room temperature (approximately 21°C), taking care not to wash or rinse the disk.
Example 2: Procedure for Isolation of Genomic DNA from Whole Blood Using Chelating Solid Phase Material One (1) pL of neat TRITON X-100 was added to one hundred (100) p,L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (for approximately S seconds every 20 seconds). The solution was investigated to make sure that it was transparent before 1 o proceeding to the next step. The solution was spun in an Eppendorf Model centrifuge (Brinkmann, Westbury, NIA at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) p.L of concentrated material at the bottom of the centrifuge tube. This material was transferred to a 3M No. 2271 EMPORE
Extraction Chelating Disk, making sure that the material was evenly distributed on the surface of the disk. The material was allowed to dry on the disk for about 2-5 minutes until the intense red color became darker. Twelve microliters (12 p,L) of 0.083 molar (M) NaOH was added to the extraction disk. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 p,L might be obtained).
The color of the sample removed from the membrane varied from colorless to faint green.
If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 p,L aliquot was removed and added to 10 p,L of 40 millimolar (mM) TRIS-HCl (Sigma-Aldrich, St. Louis, MO) (pH 7.4).
Example 3: Procedure for Isolation of Genomic DNA from Whole Blood Using Ammoniated Chelating Solid Phase Material One (1) ~.L of neat TRITON X-100 was added to one hundred (100) ~,L of whole blood. The solution was incubated at room temperature (approximately 21 °C) for about 5 minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415D
centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) p,L of concentrated material at the bottom of the centrifuge tube.
This material was transferred to the extraction membrane prepared in Example lA, making sure that the material was evenly distributed on the surface of the membrane.
The material was allowed to dry on the membrane for about 2-5 minutes until the intense red color became darker. Twelve (12) ~.L of 0.083 M NaOH was added to the extraction membrane. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 pL might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A
2 p,L
l0 aliquot was removed and added to 10 ~.L of 40 mM TRIS-HCI (pH 7.4).
Example 4: Procedure for Isolation of Genomic DNA from Whole Blood Using Ammoniated Chelating Solid Phase Material with TRITON X-100 One (1) pL of neat TRITON X-100 was added to one hundred (100) wL of whole blood. The solution was incubated at_ room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415D
centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) p.L of concentrated material at the bottom of the centrifuge tube.
This material was transferred to the extraction membrane prepared in Example 1B, making sure that the material was evenly distributed on the surface of the membrane.
The material was allowed to dry on the membrane for about 2-5 minutes until the intense red color became darker. Twelve (12) p,L of 0.083 M NaOH was added to the extraction membrane. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 ~.L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute.
The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing. A 2 p.L
aliquot was removed and added to 10 ~L of 40 mM TRIS-HCl (pH 7.4).
Example 5A: Concentration of Genomic DNA by Spinning Whole Blood Prior to Treatment with Solid Phase Material: Sample Isolation From the Top of the Tube One (1) ~,L of neat TRITON X-100 was added to one hundred (100) ~,L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415D
centrifuge at 400 rcf for about 10 minutes. A two (2) ~,L aliquot was removed from the top of the centrifuge tube and was transferred to the extraction membrane prepared in Example 1B, making sure that the material was evenly distributed on the surface of the membrane.
The material was allowed to dry on the membrane for about 2-5 minutes until the intense red color became darker. Twelve (12) ~,L of 0.083 M NaOH was added to the extraction membrane The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 ~,L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A
2 ~,L
aliquot was removed and added to 10 ~,L of 40 mM TRIS-HCl (pH 7.4).
Example 5B: Concentration of Genomic DNA by Spinning Whole Blood Prior to 2o Treatment with Solid Phase Material: Sample Isolation from the Bottom of the Tube One (1) ~L of neat TRITON X-100 was added to one hundred (100) ~,L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 541 SD
centrifuge at 400 rcf for about 10 minutes. A two (2) ~,L aliquot was removed from the bottom of the centrifuge tube and was transferred to the extraction membrane prepared in Example 1B, making sure that the material was evenly distributed on the surface of the membrane. The material was allowed to dry on the membrane for about 2-S
minutes until the intense red color became darker. Twelve (12) ~,L of 0.083 M NaOH was added to the extraction membrane. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 ~L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 p.L
aliquot was removed and added to 10 pL of 40 mM TRIS-HCl (pH 7.4).
Example SC: Purification of Genomic DNA by Treatment with Solid Phase Material:
Sample Isolation from the Top of the Tube - No Concentration Step One (1) ~L of neat TRITON X-100 was added to one hundred (100) p.L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 1o minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. A two (2) p,L aliquot was removed from the top of the centrifuge tube and was transferred to the extraction membrane prepared in Example 1B, making sure that the material was evenly distributed on the surface of the membrane. The i5 material was allowed to dry on the membrane for about 2-5 minutes until the intense red color became darker. Twelve (12) ~L of 0.083 M NaOH was added to the extraction membrane. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 ~.L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was 2o foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute.
A 2 p.L
aliquot was removed and added to 10 pL of 40 mM TRIS-HCl (pH 7.4).
Example 6A: Concentration of Genomic DNA by Spinning Whole Blood Followed by QIAamp Clean-up: Sample Isolation from the Top of the Tube 25 One (1) ~L of neat TRITON X-100 was added to one hundred (100) ~L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415D
3o centrifuge at 400 rcf for about 10 minutes. A two (2) wI, aliquot was removed from the top of the centrifuge tube and was added to 198 p,L of lx Phosphate buffer saline (PBS).
Clean DNA was obtained, following "Blood and Body Fluid Spin Protocol"
described in QIAamp DNA Blood Mini Kit Handbook p. 27(Qiagen, Valencia, CA), eluting in 72 ~,L
of water.
Example 6B: Concentration of Genomic DNA by Spinning Whole Blood Followed by QIAamp Clean-up: Sample Isolation from the Bottom of the Tube One (1) p,L of neat TRITON X-100 was added to one hundred (100) p.L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about S
minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415D
centrifuge at 400 rcf for about 10 minutes. A two (2) p,L aliquot was removed from the bottom of the centrifuge tube and was added to 198 pL of 1 x Phosphate buffer saline (PBS). Clean DNA was obtained, following "Blood and Body Fluid Spin Protocol"
described in QIAamp DNA Blood Mini Kit Handbook p. 27, eluting in 72 ~,L of water.
Example 6C: Collecttion of Genomic DNA by QIAamp Clean-up: Sample Isolation from the Top of the Tube - No Centrifuging One (1) ~,L of neat TRITON X-100 was added to one hundred (100) pL of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 s minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. A two (2) ~.L aliquot was removed from the top of the centrifuge tube and was added to 198 pL of lx Phosphate buffer saline (PBS, Sigma-Aldrich, St. Louis, MO). Clean DNA was obtained, following "Blood and Body Fluid to Spin Protocol" described in QIAamp DNA Blood Mini Kit Handbook p. 27, eluting in 72 p,L of water.
Example 7A: Effect of Inhibitor/DNA on PCR: Varying Inhibitor Concentration with Fixed DNA Concentration 15 A dilution series of inhibitors were made prior to spiking with clean human genomic DNA in order to study the effect of inhibitor on PCR. To 10 ~,L of 15 nanograms per microliter (ng/p.L) human genomic DNA, 1 wL of different Mix I (neat or dilutions thereof) was added (Samples 7 - no inhibitor added, 7D - neat, 7E - 1:10, 7F -1:30, 7G -1:100, 7H - 1:300) and vortexed. Two (2) p,L aliquots of each sample were taken for 20 ~,L
20 PCR. The results are shown in Table 2.
Mix I: one hundred (100) p,L of whole blood was added to 1 pL of neat TRITON
X-100. The solution was incubated at room temperature (approximately 21°C) for about S
minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before 25 proceeding to the next step. The solution was spun in an Eppendorf Model centrifuge at 400 rcf for about 10 minutes. Approximately 80 ~L from the top of the microcentrifuge tube and designated Mix I.
Example 7B: Effect of Inhibitor/DNA on PCR: Varying DNA Concentration with Fixed Inhibitor Concentration To 10 ~.L of human genomic DNA, 1 ~,L of 1:3 diluted Mix I (described above) was added. DNA concentrations that were examined were the following: Samples ng/~,L, 7K - 7.5 ng/wL, 7L - 3.75 ng/~L, 7M - 1.5 ngh,L. Two (2) ~L aliquots of each sample were taken for 20 ~,L PCR. The results are shown in Table 2.
Example 7C: Effect of Inhibitor/DNA on PCR: DNA with No Added Inhibitor The following samples were prepared with 1 ~,L of water added to each DNA
to sample instead of inhibitor: Samples 7N - 1 S ng/~,L, 7P - 7.5 ng/~,L, 7Q -3.75 ng/~,L, 7R -1.5 ng/~,L. Two (2) ~.L aliquots of each sample were taken for 20 ~L PCR. The results are shown in Table 2.
Table 2 Sample No. Ct (duplicateSample No. Ct (duplicate samples) samples) 7 19.10 7K 29.16 19.06 30.22 7D 13.94 7L 30.47 29.50 29.96 7E 27.39 7M 28.43 26.22 26.16 7F 21.44 7N 20.05 20.66 19.80 7G 19.90 7P 20.74 19.30 20.54 7H 19.90 7Q 21.95 20.08 21.88 7J 28.45 7R 22.67 28.61 23.10 Example 8A: Recovery of Nuclei from Solid Phase Material One (1) p.L of neat TRITON X-100 was added to 2 p,L of white blood cells (PBMCs). The solution was vortexed briefly, and was spun in an Eppendorf Model 5415D centrifuge at 400 rcf for about 1 minute. A three (3) pL was placed on a chelating membrane prepared as in Example 1B. The material was allowed to dry on the membrane for about 2-5 minutes. Thirteen (13) ~.L of 0.077 M NaOH was added to the extraction membrane. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 13 p,L might be obtained). If the solution was foamy, it to was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 p,L
aliquot was removed and added to 10 ~,L of 40 mM TRIS-HCl (pH 7.4).
Example 8B: Recovery of Nuclei with No Solid Phase Material One (1) pL of neat TRITON X-100 was added to 2 pL of white blood cells (PBMCs). The solution was vortexed briefly, and was spun in an Eppendorf Model 5415D centrifuge at 400 rcf for about 1 minute. Ten (10) p,L of 0.1 M NaOH was added to the solution and incubated at room temperature (approximately 21 °C) for about 5 minutes.
A 2 p,L aliquot was removed and added to 10 ~L of 40 mM TRIS-HCl (pH 7.4).
Example 8C: Sample Spiked Afterwards with Nuclei One (1) p,L of neat TRITON X-100 was added to 2 p,L of water. The solution was vortexed briefly, and was spun in an Eppendorf Model 5415D centrifuge at 400 rcf for about 1 minute. A three (3) ~,L was placed on a chelating membrane prepared as in Example 1B. The material was allowed to dry on the membrane for about 2-5 minutes.
Thirteen (13) ~,L of 0.077 M NaOH was added to the extraction membrane. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 13 wL might be obtained). If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. Two (2) pL of white blood cells (PBMCs) was added to a 10 p,L aliquot of the solution and incubated at room temperature (approximately 21 °C) for about S minutes. A 2 pL aliquot was removed and added to 10 ~,L of 40 mM
TRIS-HCl (pH 7.4).
Example 9A: Procedure for Isolation of Genomic DNA from Whole Blood Using Solid Phase Material: Extraction from the Solid Phase Material with NaOH
One (1) p,L of neat TRITON X-100 was added to one hundred (100) ~,L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (approximately 5 seconds every 20 seconds).
The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415D centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) pL
to of concentrated material at the bottom of the centrifuge tube. This material was transferred to the extraction membrane prepared in Example 1B, making sure that the material was evenly distributed on the surface of the membrane. The material was allowed to dry on the membrane for about 2-5 minutes until the intense red color became darker. Twelve (12) p,L of 0.083 M NaOH was added to the extraction membrane.
The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 pL might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. Fifty (50) pL of lx TE buffer (Sigma-Aldrich, St. Louis, MO) was added to a 10 p.L aliquot of the solution.
Three 2o hundred (300) p.L of 40 mM TRIS-HCl (pH 7.4) was added to the solution.
Example 9B: Procedure for Isolation of Genomic DNA from Whole Blood Using Solid Phase Material: Extraction from the Solid Phase Material with Water One (1) ~L of neat TRITON X-100 was added to one hundred (100) p,L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (approximately 5 seconds every 20 seconds).
The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415D centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) p,L
of concentrated material at the bottom of the centrifuge tube. This material was transferred to the extraction membrane prepared in Example 1B, making sure that the material was evenly distributed on the surface of the membrane. The material was allowed to dry on the membrane for about 2-5 minutes until the intense red color became darker. Twelve (12) ~.L of water was added to the extraction membrane. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 ~,L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. Fifty (50) ~.L of 0.1 M NaOH was added to a ~L aliquot of the solution and incubated at room temperature (approximately 21°C) for about S minutes. Three hundred (300) ~,L of 40 mM TRIS-HCl (pH 7.4) was added to the to solution.
Example 10: Genomic DNA Isolated from Whole Blood on Ammoniated Chelating Solid Phase Material Treated with TRITON X-100 One (1) pL of 3% TRITON X-100 was added to two (2) p,L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. This material was transferred to the extraction membrane prepared in Example 1 B, making sure that the material was evenly distributed on the surface of the membrane.
2o The material was allowed to dry on the membrane for about 2-5 minutes until the intense red color became darker. Thirteen (13) ~.L of 0.083 M NaOH was added to the extraction membrane. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing. A 2 ~L aliquot was removed and added to 10 ~L of 40 mM TRIS-HCl (pH
7.4).
Example 11: Procedure for Isolation of Genomic DNA from Whole Blood Using Anion-SR Chelating Solid Phase Material One (1) wL of neat TRITON X-100 was added to one hundred (100) pL of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 3o minutes, vortexing the solution intermittently (approximately 5 seconds every ZO seconds).
The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415 D centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) ~.L
of concentrated material at the bottom of the centrifuge tube. This material was transferred to a 3M No. 2252 EMPORE Anion-SR Extraction Chelating Disk prepared as described in Example 1C, making sure that the material was evenly distributed on the surface of the disk. The material was allowed to dry on the disk for about 2-5 minutes until the intense red color became darker. Twelve (12) ~,L of 0.083 M NaOH was added to the extraction disk. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 p.I, might be obtained). The color of the l0 sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 p,L
aliquot was removed and added to 10 p.L of 40 mM TRIS-HCl (pH 7.4).
Example 12: Procedure for Isolation of Genomic DNA from Whole Blood Using C8 Chelating Solid Phase Material One (1) ~L of neat TRITON X-100 was added to one hundred (100) p,L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (approximately 5 seconds every 20 seconds).
The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415 D centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) pL
of concentrated material at the bottom of the centrifuge tube. This material was transferred to a 3M No. 2214 EMPORE C-8 Extraction Chelating Disk prepared as described in Example 1D, making sure that the material was evenly distributed on the surface of the disk. The material was allowed to dry on the disk for about 2-5 minutes until the intense red color became darker. Twelve (12) p.L of 0.083 M NaOH was added to the extraction disk. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 ~,L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 pL
aliquot was removed and added to 10 ~,L of 40 mM TRIS-HCl (pH 7.4).
Example 13: Procedure for Isolation of Genomic DNA from Whole Blood Using Chelating Solid Phase Material One (1) wL of neat TRITON X-100 was added to one hundred (100) p,L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (approximately 5 seconds every 20 seconds).
The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415 D centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) pL
of concentrated material at the bottom of the centrifuge. tube. This material was transferred to a 3M No. 2271 EMPORE Extraction Chelating Disk prepared as described in Example lE, making sure that the material was evenly distributed on the surface of the disk. The material was allowed to dry on the disk for about 2-5 minutes until the intense red color became darker. Twelve (12) p,L of 0.083 M NaOH was added to the extraction disk. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 p,L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 ~,L aliquot was removed and added to 10 ~,L of 40 mM TRIS-HCl (pH 7.4).
Example 14: Procedure for Isolation of Genomic DNA from Whole Blood Using Chelating Solid Phase Material One (1) ~.L of neat TRITON X-100 was added to one hundred (100) p.L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (approximately 5 seconds every 20 seconds).
The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415 D centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) p,L
of concentrated material at the bottom of the centrifuge tube. This material was transferred to a 3M No. 2271 EMPORE Extraction Chelating Disk prepared as described in Example 1F, making sure that the material was evenly distributed on the surface of the disk. The material was allowed to dry on the disk for about 2-5 minutes until the intense red color became darker. Twelve (12) p.L of 0.083 M NaOH was added to the extraction disk. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 ~,L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 ~.L aliquot was removed and added to 10 p,L of 40 mM TRIS-HCl (pH 7.4).
Example 15: Procedure for Isolation of Genomic DNA from Whole Blood Using l0 Chelating Solid Phase Material One (1) pL of neat TRITON X-100 was added to one hundred (100) ~L of whole blood. The, solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (approximately 5 seconds every 20 seconds).
The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415 D centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) p,L
of concentrated material at the bottom of the centrifuge tube. This material was transferred to a ~M No. 2271 EMPORE Extraction Chelating Disk prepared as described in Example 1 G, making sure that the material was evenly distributed on the surface of the disk. The material was allowed to dry on the disk for about 2-S minutes until the intense red color became darker. Twelve (12) p,L of 0.083 M NaOH was added to the extraction disk. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 ~,L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 p,L aliquot was removed and added to 10 p.L of 40 mM TRIS-HCI (pH 7.4).
Example 16: Procedure for Isolation of Genomic DNA from Whole Blood Using Chelating Solid Phase Material 3o One (1) p.L of neat TRITON X-100 was added to one hundred (100) ~L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (approximately 5 seconds every 20 seconds).
The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415 D centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) p,L
of concentrated material at the bottom of the centrifuge tube. This material was transferred to a 3M No. 2271 EMPORE Extraction Chelating Disk prepared as described in Example 1H, making sure that the material was evenly distributed on the surface of the disk. The material was allowed to dry on the disk for about 2-5 minutes until the intense red color became darker. Twelve (12) p.L of 0.083 M NaOH was added to the extraction to disk. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 p.L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 p,L aliquot was removed and added to 10 wL of 40 mM TRIS-HCl (pH 7.4).
Example 17: Procedure for Isolation of Genomic DNA from Whole Blood Using Chelating Solid Phase Material One (1) p,L of neat TRITON X-100 was added to one hundred (100) p.L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 2o minutes, vortexing the solution intermittently (approximately 5 seconds every 20 seconds).
The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415 D centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) p,L
of concentrated material at the bottom of the centrifuge tube. This material was transferred to a 3M No. 2271 EMPORE Extraction Chelating Disk prepared as described in Example 1J, making sure that the material was evenly distributed on the surface of the disk. The material was allowed to dry on the disk for about 2-S minutes until the intense red color became darker. Twelve (12) p,L of 0.083 M NaOH was added to the extraction disk. The solution was mixed up and down 2-3 times in a pipette tip and removed after 3o mixing (although less than 12 pL might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 ~,L aliquot was removed and added to 10 pL of 40 mM TRIS-HCl (pH 7.4).
Example 18: Procedure for Isolation of Genomic DNA from Whole Blood Using Ammoniated Chelating Solid Phase Material with TRITON X-100 Mounted on a Microfluidic Device Two (2) ~.L of neat TRITON X-100 was added to one hundred (100) wL of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 l0 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415D
centrifuge at 400 rcf for about 2 min. The solution from the top was discarded leaving 4 ~,L at the bottom of the tube. To 4 p,L of concentrated material, 4 pL of RNase water was added, and the sample was mixed thoroughly. The sample then was placed into an injection port on a microfluidic device as described in Applicants' Assignee's copending U.S. Patent Application Serial No. 10/734,682, filed December 12, 2003 (Figure 1). The solution was spun at 500 rpm for 5 min to a clean up chamber containing a 3M
No. 2271 EMPORE Extraction Chelating Disk prepared as described in Example 1B using 10%
TRITON X-100 instead of 1% TRITON X-100 as a loading solution. A five (5) pL
2o aliquot of 0.1 M NaOH was added to a different injection port and was spun at 1000 rpm for 5 min to the clean up chamber described above. A ten (10) ~,L aliquot of 0.1 M NaOH
was added to the same injection port, and the solution was spun down at 2500 rpm for 10 min. A 5 p.L aliquot was removed and added to 1.5 ~,L of 1 M TRIS-HCl (pH
7.4).
Example 19: Procedure for Isolation of Genomic DNA from Whole Blood with the Use of a Chelating Solid Phase Material One hundred (100) pL of whole blood was added to one hundred (100) p.L 2%
TRITON X-100. The solution was mixed thoroughly, and then investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415D centrifuge at 400 rcf for 2 min. A 95 p.L aliquot of the solution from the top was removed and discarded. Last five (5) p,L containing concentrated material was placed onto a 3M No. 2271 EMPORE Extraction Chelating Disk prepared as described in Example 1B using 10% TRITON X-100 instead of 1% TRITON X-100 as a loading solution. After the solution had soaked into the disk, the sample was extracted with a twenty (20) pL aliquot of 0.1 M NaOH. The solution was briefly spun in an Eppendorf Model 5415D centrifuge at 400 rc~ An aliquot of eleven (11) p.L of sample was heated for 3 min at 95°C, and then added to three (3) pL of 1 M
TRIS-HCl (pH 7.4).
Example 20: Procedure for Isolation of Genomic DNA from Whole Blood Five (5) p,L of whole blood was added to the ten (10) p.L of 10 mM NaOH. After l0 min incubation, the sample was heated for 3 min at 95°C. A 5 pL
aliquot of 16 mM TRIS-HCl (pH 7.4) was added to 10 p,L of sample.
RESULTS
Table 3 reports results that were obtained on ABI 7700 QPCR Machine (Applera, 15 Foster City, CA) following the instructions in QuantiTech SYBR Green PCR
Handbook on p.10-12 for preparation of a 10 p,L PCR sample (2 p,L of sample in 10 pL
SYBR Green Master Mix, 4 p,L ~i-actin, 4 p.L of water) for Examples 1-17, 19, and 20;
Results for Example 18 were obtained on LightCycler 2.0 (Roche Applied Science, Indianapolis, IN) following the instructions in LightCycler Factor V Leiden Mutation Kit's package insert 20 on p.2-3 for preparation of a 10 p,L PCR sample (2.5 p,L of sample in 5.5 p,L of RNase-free sterile water, 1 p,L of l Ox Factor V Leiden Reaction Mix and 1 p,L of lOx Factor V Leiden Mutation Detection Mix). One (1)% agarose gel (brightness of band- + faint, +++ bright) was run on Horizon 11-14 Electrophoresis Machine (Gibco BRL, Gaithersburg, MD).
Spectra measurements were run on a SpectraMax Plus384 spectrophotometer at 405 nm 25 (Molecular Devices Corporation, Sunnyvale, CA.). Two, three or four values for each sample represent duplicates, triplicates, or quadruplicates.
Table 3 Samples Ct Band 405 nm (avg) 1.5 ng/ pL human16.92 +++ -genomic DNA in 20.67 +++
0.1 M NaOH/40mM
TRIS-HCl buffer 1.5 ng/ ~,L human19.01 +++ 0 genomic DNA in 18.67 '~++
water 1.5 ng/ p,L human16.18 +++ -genomic DNA in 16.28 +++
water Example 4 17-20 +++ 0.24 (multiple samples'T+~-analyzed) Example 3 26, 28 + -Example 2 28, 27 none -Example SA 28 + 0.37 Example SB 17,18 +++ 0.24 +++
Example SC 24 ++ -26 ++
Example 6A 23 + 0 24 +
Example 6B 17 +++ 0 18 +++
Example 6C 21 ++ 0 21 ++
Examples 7A and - - 2.63 Mix I diluted 1:36 Examples 7A and - - 0.38 Mix I diluted 1:360 Examples 7A and - - 0.036 Mix I diluted 1:3600 Examples 7A and - - 0 Mix I diluted 1:36000 Example 8A 22 +++
Example 8B 22 ++
Example 8C 20 Example 9A 21 - 0.019 19 0.007 Example 9B 25 - 0.029 28 0.074 Example 10 26.86 +++
26.11 -Example 11 24.87 - -20.38 Example 12 26.74 - -26.04 Example 13 26.01 - 0.245 Example 14 19.82 - 0.377 Example 15 27.92 30.04 Example 16 - - 0.07 Example 17 - - 0.918 Example 18 27.39 (bench - -control 27.08) Example 19* 23.72, 24.03 - -Example 20* 30.35, 30.56 - -*Positive Control for Examples 19-20 was DNA extracted from two hundred (200) p,L of whole blood following "Blood and Body Fluid Spin Protocol" described in QIAamp DNA
Blood Mini Kit Handbook p. 27, eluting in 200 p,L of water and had Ct value of 23.
Negative Control (NTC or no template control) did not amplify in these experiments.
Various modifications and alterations to this invention will become apparent to those skilled in the art without departing from the scope of this invention.
It should be understood that this invention is not intended to be unduly limited by the illustrative l0 embodiments and examples set forth herein and that such examples and embodiments are presented by way of example only with the scope of the invention intended to be limited only by the claims set forth herein as follows.
International Publication No. WO 01/37291 A1 (MagNA Pure) describes the use of magnetic glass particles and an isolation method in which samples are lysed by incubation with a special buffer containing a chaotropic salt and proteinase K. Glass magnetic particles are added and total nucleic acids contained in the sample are bound to their surface. Unbound substances are removed by several washing steps. Finally, purified total nucleic acid is eluted with a low salt buffer at high temperature.
Yet another conventional method involves applying a biological sample to a hydrophobic organic polymeric solid phase to selectively trap nucleic acid and subsequently remove the trapped nucleic acid with a nonionic surfactant.
Another method involves treating a hydrophobic organic polymeric material with a nonionic surfactant, washing the surface, and subsequently contacting the treated solid organic polymeric material with a biological sample to reduce the amount of nucleic acid that binds to the organic polymeric solid phase. Although these solid phase methods are effective methods for isolating nucleic acid from biological samples, other methods are needed, particularly methods that are suitable for use in microfluidic devices.
The discussion of prior publications and other prior knowledge does not constitute an admission that such material was published, known, or part of the common general knowledge.
SUMMARY
The present invention provides methods for the isolation, and preferably purification and recovery, of nucleic acids.
Nucleic acids isolated according to the invention, will be useful, for example, in assays for detection of the presence of a particular nucleic acid in a sample.
Such assays are important in the prediction and diagnosis of disease, forensic medicine, epidemiology, and public health. For example, isolated DNA may be subjected to hybridization and/or amplification to detect the presence of an infectious virus or a mutant gene in an individual, allowing determination of the probability that the individual will suffer from a disease of infectious or genetic origin. The ability to detect an infectious virus or a mutation in one sample among the hundreds or thousands of samples being screened takes on substantial importance in the early diagnosis or epidemiology of an at-risk population for disease, e.g., the early detection of HIV infection, cancer or susceptibility to cancer, or in the screening of newborns for diseases, where early detection may be instrumental in diagnosis and treatment. In addition, the methods of the present invention can also be used in basic research laboratories to isolate nucleic acid from cultured cells or biochemical reactions. The nucleic acid can be used for enzymatic modification such as restriction enzyme digestion, sequencing, and amplification.
1o The present invention provides methods and kits for isolating nucleic acid from a sample that includes nucleic acid (e.g., DNA, RNA, PNA), which may or may not be included within nuclei-containing cells (e.g., white blood cells). These methods involve ultimately separating nucleic acid from inhibitors, such as heme and degradation products thereof (e.g., iron ions or salts thereof), which are undesirable because they can inhibit amplification reactions (e.g., as are used in PCR reactions).
Certain embodiments of the invention involve retaining inhibitors in or on a solid phase material (i.e., adhering the inhibitors to the material) without retaining a significant amount of nucleic acid. Suitable solid phase materials typically .include a solid matrix in any form (e.g., particles, fibrils, a membrane) with capture sites (e.g., chelating functional groups) attached thereto, a coating reagent (preferably, a surfactant) coated on the solid phase material, or both.
In one embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method including: providing a sample including nucleic acid-containing material and inhibitors (typically, contained in cells; such nucleic acid-containing material and cells containing inhibitors may be the same or different); if the sample includes cells containing inhibitors, the method includes optionally contacting the sample with a first lysing reagent under conditions effective to break cell merribranes and release inhibitors and form a lysed sample including nucleic acid-containing material and inhibitors; forming a concentrated region of the sample (typically, lysed sample); wherein the concentrated region of the sample (typically, lysed sample) includes nucleic acid-containing material and inhibitors; substantially separating the concentrated region from a less concentrated region of the sample (typically, lysed sample); contacting the separated concentrated region of the sample (typically, lysed sample) with a solid phase material to preferentially adhere at least a portion of the inhibitors (i.e., at least a portion of at least one inhibitor) to the solid phase material, wherein the solid phase material includes a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barner, and combinations thereof; optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material;
and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method including: providing a sample including cells containing inhibitors and cells containing nuclei (such cells containing inhibitors and cells containing nuclei may be the same or different); contacting the biological sample with a nonionic surfactant under conditions effective to break cell membranes and release nuclei and inhibitors and form a lysed sample; forming a concentrated region of the lysed sample;
wherein the concentrated region of the sample includes nuclei and inhibitors;
substantially separating the concentrated region of the lysed sample from the less concentrated region of the sample; contacting the separated concentrated region of the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material includes a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof; optionally further lysing the nuclei to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nucleic acid-containing and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method including: providing a microfluidic device including a loading chamber, a valued process chamber, and a separation chamber including a solid phase material; providing a sample including nucleic acid-containing material and inhibitors (typically, contained in cells; such nucleic acid-containing material and cells containing inhibitors may be the same or different); placing the sample in the loading chamber; if the sample includes cells containing inhibitors, optionally contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample including nucleic acid-containing material and inhibitors; transfernng the sample (typically, lysed sample) to a valued process chamber; forming a concentrated region of the sample (typically, lysed sample) in the valued process chamber, wherein the concentrated region of the sample (typically, lysed sample) includes nucleic acid-containing material and inhibitors;
substantially separating the concentrated region from a less concentrated region of the sample (typically, lysed sample); transferring the separated concentrated region of the sample (typically, lysed sample) to the separation chamber for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material includes capture sites (e.g., chelating functional groups), a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof; optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material;
and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method including: providing a microfluidic device including a loading chamber, a valued process chamber, and a separation chamber including a solid phase material; providing a sample including cells containing inhibitors and cells containing nuclei (such cells containing inhibitors and cells containing nuclei may be the same or different); placing the sample in the loading chamber;
contacting the sample with a nonionic surfactant under conditions effective to break cell membranes and release nuclei and inhibitors to form a lysed sample; transferring the lysed sample to a valued process chamber; forming a concentrated region of the lysed sample in the valued process chamber, wherein the concentrated region of the lysed sample includes nuclei and inhibitors; substantially separating the concentrated region from a less concentrated region of the lysed sample; transferring the separated concentrated region of the lysed sample to the separation chamber for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material includes capture sites (e.g., chelating functional groups), a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof; optionally further lysing the nuclei to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the i5 nuclei and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method including: providing a sample including nucleic acid and inhibitors; contacting the sample with a solid phase material to preferentially 2o adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material includes a polytetrafluoroethylene fibril matrix, soiptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barner, and combinations thereof;
25 optionally lysing nucleic acid-containing material, if present, to release nucleic acid before, simultaneous with, or after contacting the lysed sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic 30 surfactant.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method including: providing a sample including nucleic acid-containing material (e.g., nuclei) and inhibitors (typically, contained in cells; such nucleic acid-containing material and cells containing inhibitors may be the same or different); if the sample includes cells containing inhibitors, optionally contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample including nucleic acid-containing material and inhibitors; contacting the sample (typically, lysed sample) with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material includes a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof (preferably, a surfactant); optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the lysed sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic surfactant.
2o In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method including: providing a sample including nucleic acid-containing material (e.g., nuclei) and cells containing inhibitors (which may be the same or different); contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and for~rr a lysed sample including nucleic acid-containing material and inhibitors; contacting the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material includes a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, 3o a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof (preferably, a surfactant); separating at least a portion of the nucleic acid-containing material from the solid phase material having at least a portion of the inhibitors adhered thereto; and after separating the nucleic acid-containing material, further lysing the nucleic acid-containing material to release nucleic acid.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method including: providing a microfluidic device including a loading chamber and a separation chamber including a solid phase material;
providing a sample including nucleic acid-containing material and inhibitors (typically, contained in cells; such nucleic acid-containing material and cells containing inhibitors may be the same or different); placing the sample in the loading chamber; if the sample includes cells containing inhibitors, optionally contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample including nucleic acid-containing material and inhibitors;
transferring the sample (typically, lysed sample) to the separation chamber to contact the solid phase material and to preferentially adhere at least a portion of the inhibitors to the solid phase material; optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the sample with the solid phase material; wherein the solid phase material includes capture sites (e.g., chelating functional groups), a coating reagent coated on the solid phase material, or both;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barner, and combinations thereof; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic surfactant.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method including: providing a microfluidic device including a loading chamber and a separation chamber including a solid phase material;
providing a sample including nucleic acid-containing material and cells containing inhibitors (which may be the same or different); placing the sample in the loading 3o chamber; contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample including nucleic acid-containing material and inhibitors; transferring the lysed sample to the separation chamber to contact the solid phase material and to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material includes capture sites (e.g., chelating functional groups), a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barner, and combinations thereof; and separating at least a portion of the nucleic acid-containing material from the solid phase material having at least a portion of the inhibitors adhered thereto; and after separating the nucleic acid-containing material, further lysing the nucleic l0 acid-containing material to release nucleic acid.
The present invention also provides kits for carrying out the various methods of the present invention.
DEFINITIONS
"Nucleic acid" shall have the meaning known in the art and refers .to DNA
(e.g., genomic DNA, cDNA, or plasmid DNA), RNA (e.g., mRNA, tRNA, or rRNA), and PNA.
It can be in a wide variety of forms, including, without limitation, double-stranded or single-stranded configurations, circular form, plasmids, relatively short oligonucleotides, peptide nucleic acids also called PNA's (as described in Nielsen et al., Chem.
Soc. Rev., 26, 73-78 (1997)), and the like. The nucleic acid can be genomic DNA, which can include an entire chromosome or a portion of a chromosome. The DNA can include coding (e.g., for coding mRNA, tRNA, and/or rRNA) and/or noncoding sequences (e.g., centromeres, telomeres, intergeriic regions, introns, transposons, and/or microsatellite sequences). The nucleic acid can include any of the naturally occurnng nucleotides as well as artificial or chemically modified nucleotides, mutated nucleotides, etc. The nucleic acid can include a non-nucleic acid component, e.g., peptides (as in PNA's), labels (radioactive isotopes or fluorescent markers), and the like.
"Nucleic acid-containing material" refers to a source of nucleic acid such as a cell (e.g., white blood cell, enucleated red blood cell), a nuclei, or a virus, or any other composition that houses a structure that includes nucleic acid (e.g., plasmid, cosmid, or viroid, archeobacteriae). The cells can be prokaryotic (e.g., gram positive or gram negative bacteria) or eukaryotic (e.g., blood cell or tissue cell). If the nucleic acid-containing material is a virus, it can include an RNA or a DNA genome; it can be virulent, attenuated, or noninfectious; and it can infect prokaryotic or eukaryotic cells. The nucleic acid-containing material can be naturally occurnng, artificially modified, or artificially created.
"Isolated" refers to nucleic acid (or nucleic acid-containing material) that has been separated from at least a portion of the inhibitors (i.e., at least a portion of at least one inhibitor) in a sample. This includes separating desired nucleic acid from other materials, e.g., cellular components such as proteins, lipids, salts, and other inhibitors. More 1o preferably, the isolated nucleic acid is substantially purified.
"Substantially purified"
refers to isolating nucleic acid of at least 3 picogram per microliter (pg/p,L), preferably at least 2 nanogram/microliter (ng/~,L), and more preferably at least 15 ng/p,L, while reducing the inhibitor amount from the original sample by at least 20%, preferably by at least 80%
and more preferably by at least 99%. The contaminants are typically cellular components and nuclear components such as heme and related products (hemin, hematin) and metal ions, proteins, lipids, salts, etc., other than the solvent in the sample.
Thus, the term "substantially purified" generally refers to separation of a majority of inhibitors (e.g., heme and it degradation products) from the sample, so that compounds capable of interfering with the subsequent use of the isolated nucleic acid are at least partially removed.
"Adheres to" or "adherence" or "binding" refer to reversible retention via a wide variety of mechanisms, including weak forces such as Van der Waals interactions, electrostatic interactions, affinity binding, or physical trapping. The use of this term does not imply a mechanism of action, and includes adsorptive and absorptive mechanisms.
"Solid phase material" refers to an inorganic and/or organic material, preferably a polymer made of repeating units, which may be the same or different, of organic and/or inorganic compounds of natural and/or synthetic origin. This includes homopolymers and heteropolymers (e.g., copolymers, terpolymers, tetrapolymers, etc., which may be random or block, for example). This term includes fibrous or particulate forms of a material, which can be readily prepared by methods well-known in the art. Such materials typically form a porous matrix, although for certain embodiments, the solid phase also refers to a solid surface, such as a nonporous sheet of polymeric material.
The solid phase material may include capture sites. "Capture sites" refer to sites on the solid phase material to which a material adheres. Typically, the capture sites include functional groups or molecules that are either covalently attached or otherwise attached (e.g., hydrophobically attached) to the solid phase material.
The phrase "coating reagent coated on the solid phase material" refers to a material coated on at least a portion of the solid phase material, e.g., on at least a portion of the fibril matrix and/or sorptive particles.
"Surfactant" refers to a substance that lowers the surface or interfacial tension of the medium in which it is dissolved.
"Strong base" refers to a base that is completely dissociated in water, e.g., NaOH.
"Polyelectrolyte" refers to an electrolyte that, is a charged polymer, typically of relatively high molecular weight, e.g., polystyrene sulfonic acid.
"Selectively permeable polymeric barrier" refers to a polymeric barrier that allows for selective transport of a fluid based on size and charge.
"Concentrated region" refers to a region of a sample that has a higher concentration of nucleic acid-containing material, nuclei, and/or nucleic acid, which can be in a pellet form, relative to the less concentrated region.
"Substantially separating" as used herein, particularly in the context of separating a concentrated region of a sample from a less concentrated region of a sample, means 2o removing at least 40% of the total amount of nucleic acid (whether it be free, within nuclei, or within other nucleic acid-containing material) in less than 25% of the total volume of the sample. Preferably, at least 75% of the total amount of nucleic acid in less than 10% of the total volume of sample is separated from the remainder of the sample.
More preferably, at least 95% of the total amount of nucleic acid in less than 5% of the total volume of sample is separated from the remainder of the sample.
"Inhibitors" refer to inhibitors of enzymes used in amplification reactions, for example. Examples of such inhibitors typically include iron ions or salts thereof (e.g., Fe2+ or salts thereof) and other metal salts (e.g., alkali metal ions, transition metal ions).
Other inhibitors can include proteins, peptides, lipids, carbohydrates, heme and its 3o degradation products, urea, bile acids, humic acids, polysaccharides, cell membranes, and cytosolic components. The major inhibitors in human blood for PCR are hemoglobin, lactofernn, and IgG, which are present in erythrocytes, leukocytes, and plasma, respectively. The methods of the present invention separate at least a portion of the inhibitors (i.e., at least a portion of at least one type of inhibitor) from nucleic acid-containing material. As discussed herein, cells containing inhibitors can be the same as the cells containing nuclei or other nucleic acid-containing material.
Inhibitors can be contained in cells or be extracellular. Extracellular inhibitors include all inhibitors not contained within cells, which includes those inhibitors present in serum or viruses, for example.
"Preferentially adhere at least a portion of the inhibitors to the solid phase material"
1o means that one or more types of inhibitors will adhere to the solid phase material to a greater extent than nucleic acid-containing material (e.g., nuclei) and/or nucleic acid, and typically without adhering a substantial portion of the nucleic acid-containing material and/or nuclei to the solid phase material.
"Microfluidic" refers to a device with one or more fluid passages, chambers, or 15 conduits that have at least one internal cross-sectional dimension, e.g., depth, width, length, diameter, etc., that is less than 500 pm, and typically between 0.1 p,m and 500 p,m.
In the devices used in the present invention, the microscale channels or chambers preferably have at least one cross-sectional dimension between 0.1 p,m and 200 p,m, more preferably between 0.1 pm and 100 pm, and often between 1 p,m and 20 p.m.
Typically, a 2o microfluidic device includes a plurality of chambers (process chambers, separation chambers, mixing chambers, waste chambers, diluting reagent chambers, amplification reaction chambers, loading chambers, and the like), each of the chambers defining a volume for containing a sample; and at least one distribution channel connecting the plurality of chambers of the array; wherein at least one of the chambers within the array 25 can include a solid phase material (thereby often being referred to as a separation chamber) and/or at least one of the process chambers within the array can include a lysing reagent (thereby often being referred to as a mixing chamber), for example.
The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.
As used herein "a " "an " "the " "at least one " and "one or more" are used , , , , , interchangeably and mean one or more.
Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
Furthermore, various embodiments are described in which the various elements of each embodiment could be used in other embodiments, even though not specifically described.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1-2 are representations of microfluidic devices used in certain methods of the present invention.
2o DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention provides various methods and kits for isolating nucleic acid from a sample, typically a biological sample, preferably in a substantially purified form.
The present invention provides methods and kits for isolating nucleic acid from a sample that includes nucleic acid (e.g., DNA, RNA, PNA), which may or may not be included within nuclei-containing cells (e.g., white blood cells).
It should be understood that although the methods are directed to isolating nucleic acid from a sample, the methods do not necessarily remove the nucleic acid from the nucleic acid-containing material (e.g., nuclei). That is, further steps may be required to further separate the nucleic acid from the nuclei, for example.
The methods of the present invention involve ultimately separating nucleic acid from inhibitors, such as heme and degradation products thereof (e.g., iron salts), which are undesirable because they can inhibit amplification reactions (e.g., as are used in PCR
reactions). More specifically, the methods of the present invention involve separating at least a portion of the nucleic acid in a sample from at least a portion of at least one type of inhibitor. Preferred methods involve removing substantially all the inhibitors in a sample containing nucleic acid such that the nucleic acid is substantially pure. For example, the final concentration of iron-containing inhibitors is no greater than about 0.8 micromolar (p,M), which is the current level tolerated in conventional PCR systems.
In order to get clean DNA from whole blood, removal of hemoglobin as well as plasma proteins is typically desired. When red blood cells are lysed, heme and related to compounds are released that inhibit Taq Polymerise. The normal hemoglobin concentration in whole blood is 15 grams (g) per 100 milliliters (mL) based on which the concentration of heme in hemolysed whole blood is around 10 millimolar (mM).
For PCR
to work out satisfactorily, the concentration of heme should be reduced to the micromolar (pM) level. This can be achieved by dilution or by removal of inhibitors using a material that binds inhibitors, for example.
Typically, a sample containing nucleic acid is processed in a flow-through receptacle, although this receptacle is not a necessary requirement of the present invention.
Preferably, for certain methods of the present invention, the processing equipment is in a microfluidic format.
SAMPLES
The methods of the present invention can be used to isolate nucleic acids from a wide variety of samples, particularly biological samples, such as body fluids (e.g., whole blood, blood serum, urine, saliva, cerebral spinal fluid, semen, or synovial lymphatic fluid), various tissues (e.g., skin, hair, fur, feces, tumors, or organs such as liver or spleen), cell cultures or cell culture supernatants, etc. The sample can be a food sample, a beverage sample, a fermentation broth, a clinical sample used to diagnose, treat, monitor, or cure a disease or disorder, a forensic sample, an agricultural sample (e.g., from a plant or animal), or an environmental sample (e.g., soil, dirt, or garbage).
3o Biological samples are those of biological or biochemical origin. Those suitable for use in the methods of the present invention can be derived from mammalian, plant, bacterial, or yeast sources. The biological sample can be in the form of single cells or in the form of a tissue. Cells or tissue can be derived from in vitro culture.
Significantly, certain embodiments of the invention use whole blood without any preprocessing (e.g., lysing, filtering, etc.) as the sample of interest.
For certain embodiments, a sample such as whole blood can be preprocessed by centrifuging and the white blood cells (i.e., the huffy coat) separated from the blood and used as the sample in the methods of the invention.
For certain embodiments, a sample can be subjected to ultracentrifugation to concentrate the sample prior to subjecting it to a process of the present invention.
to The sample can be a solid sample (e.g., solid tissue) that is dissolved or dispersed in water or an organic medium, or from which the nucleic acid has been extracted into water or an organic medium. For example, the sample can be an organ homogenate (e.g., liver, spleen). Thus, the sample can include previously extracted nucleic acid (particularly if it is a solid sample).
The type of sample is not a limitation of the present invention. Typically, however, the sample will include nucleic acid-containing material and inhibitors from which the nucleic acid needs to be separated. In this context, nucleic acid-containing material refers to cells (e.g., white blood cell; bacterial cells), nuclei, viruses, or any other composition that houses a structure that includes nucleic acid (e.g., plasmid, cosmid, or viroid, archeobacteriae). In certain preferred embodiments of such methods, the nucleic acid-containing material includes nuclei. In certain embodiments, such nuclei are in tact (i.e., substantially unlysed) when they contact the solid phase material described herein.
1n certain embodiments, the sample may be partially lysed (e.g., pre-lysed to release inhibitors), in which case lysing may be required in the process of the present invention, or fully lysed. Thus, a sample can include free (e.g., not within cells) nucleic acid and free (e.g., not within cells) inhibitors.
The isolated (i.e., separated from inhibitors) nucleic acid can be used, preferably without further purification or washing, for a wide variety of applications (e.g., amplification, sequencing, labeling, annealing, restriction digest, ligation, reverse 3o transcriptase, hybridization, Southern blot, Northern blot, etc.). In particularly, it can be used for determining a subject's genome. It can be used for the diagnosis of the presence of a microorganism (e.g., bacteria, virus) in a sample, and subsequently can be used for monitoring and/or remedying the damage caused by the microorganism to the source of the sample. The methods, materials, systems, and kits of the present invention are especially well-suited for preparing nucleic acid extracts for use in amplification techniques (e.g., PCR, LCR, MASBA, SDA, and bDNA) used in high throughput or automated processes, particularly microfluidic systems. Thus, for certain embodiments of the present invention, the isolated nucleic acid is transferred to an amplification reaction chamber (such as a PCR
sample chamber in a microfluidic device).
The nucleic acids may be isolated (i.e., separated from inhibitors) according to the 1o invention from an impure, partially pure, or a pure sample. The purity of the original sample is not critical, as nucleic acid may be isolated from even grossly impure samples.
For example, nucleic acid may be obtained from an impure sample of a biological fluid such as blood, saliva, or tissue. If an original sample of higher purity is desired, the sample may be treated according to any conventional means known to those of skill in the art prior to undergoing the methods of the present invention. For example, the sample may be processed so as to remove certain impurities such as insoluble materials prior to subjecting the sample to a method of the present invention.
The nucleic acid isolated as described herein may be of any molecular weight and in single-stranded form, double-stranded form, circular, plasmid, etc. Various types of 2o nucleic acid can be separated from each other (e.g., RNA from DNA, or double-stranded DNA from single-stranded DNA). For example, small oligonucleotides or nucleic acid molecules of about 10 to about 50 bases in length, much longer molecules of about 1000 bases to about 10,000 bases in length, and even high molecular weight nucleic acids of about 50 kb to about 500 kb can be isolated using the methods of the present invention. In some aspects, a nucleic acid isolated according to the invention may preferably be in the range of about 10 bases to about 100 kilobases.
The nucleic acid-containing sample may be in a wide variety of volumes. For example, the applied volume may be as large as 1 liter or as small as 1 ~L, or even less.
The sample size typically varies depending on the equipment used to carry out the method.
For a microfluidic format, typically very small volumes, e.g., 10 p.L (and preferably, no greater than 100 p.L) are preferred. It should be understood that larger samples can be used if preprocessed, such as by concentrating.
For low copy number genes, one typically would need a larger sample size to ensure that the sequence of interest is present in the sample. Larger sample sizes, however, have a greater amount of inhibitors and do not typically lend themselves to a microfluidic format. Thus, for a low copy number situation, it may be necessary to use a 100 p,L or higher volume in order to get a reproducible result; however, the number of samples processed per microfluidic device may be reduced due to the higher sample volume.
l0 In the methods of the present invention, a centrifugation step to concentrate nucleic acid-containing material is useful for low copy number samples. However, while the nucleic acid concentration is increased substantially at the bottom of the process chamber, the inhibitor concentration is still high. While most of the inhibitors, the proteins in the serum and the broken RBC's (e.g., heme and heme-related products) are removed in the is supernatant; the nucleic acid-containing concentrated region of the sample still has a significant amount of inhibitor present; however, the ratio of nucleic acid to that of the inhibitor is very high, resulting in an enriched sample with respect to nucleic acid. This concentrated region of the sample can then be contacted with a solid phase material, as described herein, to remove residual inhibitors.
20 For high copy number genes, a sample size as small as 2 ~L can be used, but reproducibility is better with larger volumes (e.g., 20 pL). In the case of smaller volumes, higher throughputs (i.e., number of samples processed per microfluidic device) can be obtained. In the case of larger volumes (e.g., 20 pL), it may not be necessary to go through a pre-spin step for concentration of nucleic acid-containing cells.
25 For certain embodiments, the nucleic acid-containing sample applied to the solid phase material may be any amount, that amount being determined by the amount of the solid phase material. Preferably, the amount of nucleic acid in a sample applied to the solid phase material is less than the dried weight of the solid phase material, typically about 1/10,000 to about 1/100 (weight nucleic acid/solid phase). The amount of nucleic 30 acid in a sample applied to the solid phase material may be as much as 100 grams or as little as 1 picogram, for example.
The desired nucleic acid isolated from the methods of the present invention is preferably in an amount of at least 20%, more preferably in an amount of at least 30%, more preferably at least 70%, and most preferably at least 90%, of the amount of total nucleic acid in the originally applied sample. Thus, certain preferred methods of the present invention provide for high recovery of the desired nucleic acid from a sample.
Furthermore, exceedingly small amounts of nucleic acid molecules may be quantitatively recovered according to the invention. The recovery or yield is mainly dependent on the quality of the sample rather than the procedure itself. Because certain embodiments of the invention provide a nucleic acid preparation that does not require concentration from a to large volume, the invention avoids risk of loss of the nucleic acid.
Having too much DNA in a PCR sample can be detrimental to amplification of DNA as there are a lot of misprimed sites. This results in a large number of linearly or exponentially amplified non-target sequences. Since the specificity of the amplification is lost as the amount of non-target DNA is increased, the exponential accumulation of the target sequence of interest does not occur to any significant degree. Thus, it is desirable to control the amount of DNA that goes into each PCR sample. The DNA amount is typically not more than 1 microgram/reaction, typically at least 1 picogram/reaction.
The typical final DNA concentration in a PCR mixture ranges from 0.15 nanogram/microliter to 1.5 nanograms/microliter. In the case of a microfluidic device, a sample can be split after 2o clean-up, prior to PCR, such that each sample has the right amount of DNA.
Alternatively, a sample can be diluted sufficiently in a sample processing device (particularly, a microfluidic device) that includes a variable valued process chamber, described in greater detail below, so that the right amount of DNA is present in each PCR
mixture. 1n a diagnostic setting, since the amount of white blood cells can vary significantly, it is hard to apriori predict the amount of DNA that will be isolated.
However, a useful range is 3 micrograms (p.g) to 12 pg of DNA per 200 pL of blood. For buffy coats, 25 pg to 50 pg per 200 pL of buffy coat is a useful range.
LYSING REAGENTS AND CONDITIONS
3o For certain embodiments of the invention, at some point during the process, cells within the sample, particularly nucleic acid-containing cells (e.g., white blood cells, bacterial cells, viral cells) are lysed to release the contents of the cells and form a sample (i.e., a lysate). Lysis herein is the physical disruption of the membranes of the cells, refernng to the outer cell membrane and, when present, the nuclear membrane.
This can be done using standard techniques, such as by hydrolyzing with proteinases followed by heat inactivation of proteinases, treating with surfactants (e.g., nonionic surfactants or sodium dodecyl sulfate), guanidinium salts, or strong bases (e.g., NaOH), disrupting physically (e.g., with ultrasonic waves), boiling, or heating/cooling (e.g., heating to at least 55°C (typically to 95°C) and cooling to room temperature or below (typically to 8°C)), which can include a freezing/thawing process. Typically, if a lysing reagent is used, it is in to aqueous media, although organic solvents can be used, if desired.
Lysing of red blood cells (RBC's) without the destruction of white blood cells (WBC's) in whole blood can occur to release inhibitors through the use of water (i.e., aqueous dilution) as the lysing agent (i.e., lysing reagent). Alternatively, ammonium chloride or quaternary ammonium salts can also be used to break RBC's. The RBC's can also be lysed by hypotonic shock with the use of a hypotonic buffer. The in-tact WBC's or their nuclei can be recovered by centrifugation, for example.
Typically, a stronger lysing reagent, such as a surfactant, can be used to lyse RBC's as well as nucleic acid-containing cells (e.g., white blood cells (WBC's), bacterial cells, viral cells) to release inhibitors, nuclei, and/or nucleic acid. For example, a nonionic surfactant can be used to lyse RBC's as well as WBC's while leaving the nuclei in tact.
Nonionic surfactants, cationic surfactants, anionic surfactants, and zwitterionic surfactants can be used to lyse cells. Particularly useful are nonionic surfactants.
Combinations of surfactants can be used if desired. A nonionic surfactant such as TRITON X-100 can be added to a TRIS buffer containing sucrose and magnesium salts for isolation of nuclei.
The amount of surfactant used for lysing is sufficiently high to effectively lyse the sample, yet sufficiently low to avoid precipitation, for example. The concentration of surfactant used in lysing procedures is typically at least 0.1 wt-%, based on the total weight of the sample. The concentration of surfactant used in lysing procedures is typically no greater than 4.0 wt-%, and preferably, no greater than 1.0 wt-%, based on the total weight of the sample. The concentration is usually optimized in order to obtain complete lysis in the shortest possible time with the resulting mixture being PCR compatible. In fact, the nucleic acid in the formulation added to the PCR cocktail should allow for little or no inhibition of real-time PCR.
If desired, a buffer can be used in admixture with the surfactant. Typically, such buffers provide the sample with a pH of at least 7, and typically no more than 9.
Typically, an even stronger lysing reagent, such as a strong base, can be used to lyse any nuclei contained in the nucleic acid-containing cells (as in white blood cells) to release nucleic acid. For example, the method described in U.S. Pat. No.
5,620,852 (Lin et al.), which involves extraction of DNA from whole blood with alkaline treatment (e.g., NaOH) at room temperature in a time frame as short as 1 minute, can be adapted to certain l0 methods of the present invention. Generally, a wide variety of strong bases can be used to create an effective pH (e.g., 8-13, preferably 13) in an alkaline lysis procedure. The strong base is typically a hydroxide such as NaOH, LiOH, KOH; hydroxides with quaternary nitrogen-containing cations (e.g., quaternary ammonium) as well as bases such as tertiary, secondary or primary amines. Typically, the concentration of the strong base is at least 15 0.01 Normal (I~, and typically, no more than 1 N. Typically, the mixture can then be neutralized, particularly if the nucleic acid is subjected to PCR. In another procedure, .
heating can be used subsequent to lysing with base to further denature proteins followed by neutralizing the sample.
One can also use Proteinase K with heat followed by heat inactivation of proteinase 20 K at higher temperatures for isolation of nucleic acids from the nuclei or WBC.
One can also use a commercially available lysing agent and neutralization agent such as in Sigma's Extract-N-Amp Blood PCR kit scaled down to microfluidic dimensions. Stonger lysing solutions such as POWERLYSE from GenPoint (Oslo, Norway) for lysing difficult bacteria such as Staphylococcus, Streptococcus, etc., can be 25 used to advantage in certain methods of the present invention.
In another procedure, a boiling method can be used to lyse cells and nuclei, release DNA, and precipitate hemoglobin simultaneously. The DNA in the supernatant can be used directly for PCR without a concentration step, making this procedure useful for low copy number samples.
3o For infectious diseases, it may be necessary to analyze bacterial or viruses from whole blood. For example, in the case of bacteria, white blood cells may be present in conjunction with bacterial cells. In methods that use a microfluidic device, it would be possible to lyse red blood cells to release inhibitors, and then separate out bacterial cells and white blood cells by centrifugation, for example, prior to further lysing.
This concentrated slug of nucleic acid-containing cells (bacterial and white blood cells/nuclei) can be moved further into a chamber containing the solid phase material for removal of inhibitors. Then, the bacterial cells, for example, can be lysed.
Bacterial cell lysis, depending on the type, may be accomplished using heat.
Alternatively, bacterial cell lysis can occur using enzymatic methods (e.g., lysozyme, mutanolysin) or chemical methods. The bacterial cells are preferably lysed by alkaline lysis.
The use of bacteria for propagation of plasmids is common in the study of genomics, analytic molecular biology, preparatory molecular biology, etc. In the case of the bacterium containing plasmid, genetic material from both the bacterium and the plasmid are present. A clean-up procedure to separate cellular proteins and cellular fragments from genomic DNA can be carried out using a method of the present invention.
The supernatant thus obtained, which contains the plasmid DNA, is called the "cleared lysate." The cleared lysate can be further purified using a variety of means, such as anion-exchange chromatography, gel filtration, or precipitation with alcohol.
In a specific example of a protocol for bacterial cultures, which can be incorporated into a microfluidic device, an E. Coli cell culture is centrifuged and resuspended in TE
buffer (10 mM TRIS, 1 mM EDTA, pH 7.5) and lysed by the addition of 0.1 M
NaOH/1%
SDS (sodium dodecyl sulfate). The cell lysis is stopped by the addition of 1 volume of 3 M (three molar) potassium acetate (pH 4.8) and the supernatant centrifuged.
The cell lysate is further purified to get clean plasmid DNA.
Plasma and serum represent the majority of specimens submitted for molecular testing that include viruses. After fractionation of whole blood, plasma or serum samples can be used for the extraction of viruses (i.e., viral particles). For example, to isolate DNA
from viruses, it is possible in the microfluidic case, to first separate out the serum by spinning blood. By the use of the variable valve, which is described in greater detail below, the serum alone can be emptied into another chamber. The serum can then be centrifuged to concentrate the virus or can be used directly in subsequent lysis steps.after removal of the inhibitors using a solid phase material, for example, as described herein.
The solid phase material could absorb the solution such that the virus particles do not go through the material. The virus particles can then be eluted out in a small elution volume.
The virus can be lysed by heat or by enzymatic or chemical means, for example, by the use of surfactants, and used for downstream applications, such as PCR or real-time PCR. In cases where viral RNA is required, it may be necessary to have an RNAse inhibitor added to the solution to prevent degradation of RNA.
SOLID PHASE MATERIAL
For certain embodiments of the invention, it has been found that inhibitors will adhere to solid phase (preferably polymeric) materials that include a solid matrix in any form (e.g., particles, fibrils, a membrane), preferably with capture sites (e.g., chelating functional groups) attached thereto, a coating reagent (preferably, surfactant) coated on the solid phase material (i.e., at least a portion thereof), or both. The coating reagent can be a cationic, anionic, nonionic, or zwitterionic surfactant. Alternatively, the coating reagent can be a polyelectrolyte, a strong base, or a selectively permeable polymeric barner.
Various combinations of coating reagents can be used if desired.
The solid phase material useful in the methods of the present invention may include a wide variety of organic and/or inorganic materials that retain inhibitors such as heme and heme degradation products, particularly iron ions, for example. Such materials are functionalized with capture sites (preferably, chelating groups), coated with one or more coating reagents (e.g., surfactants, polyelectrolytes, or strong bases), or both.
Typically, the solid phase material includes an organic polymeric matrix.
Generally suitable materials are chemically inert, physically and chemically stable, and compatible with a variety of biological samples. Examples of solid phase materials include silica, zirconia, alumina beads, metal colloids such as gold, gold coated sheets that have been functionalized through mercapto chemistry, for example, to generate capture sites. Examples of suitable polymers include for example, polyolefins and fluorinated polymers. The solid phase material is typically washed to remove salts and other contaminants prior to use. It can either be stored dry or in aqueous suspension ready for use. The solid phase material is preferably used in a flow-through receptacle, for example, such as a pipet, syringe, or larger column, microtiter plate, or microfluidic device, although suspension methods that do not involve such receptacles could also be used.
The solid phase material useful in the methods of the present invention can include a wide variety of materials in a wide variety of forms. For example, it can be in the form of particles or beads, which may be loose or immobilized, fibers, foams, frits, microporous film, membrane, or a substrate with microreplicated surface(s). If the solid phase material includes particles, they are preferably uniform, spherical, and rigid to ensure good fluid flow characteristics.
For flow-through applications of the present invention, such materials are typically 1o in the form of a loose, porous network to allow uniform and unimpaired entry and exit of large molecules and to provide a large surface area. Preferably, for such applications, the solid phase material has a relatively high surface area, such as, for example, more than one meter squared per gram (mz/g). For applications that do not involve the use of a flow-through device, the solid phase material may or may not be in a porous matrix.
Thus, membranes can also be useful in certain methods of the present invention.
For applications that use particles or beads, they may be introduced to the sample or the sample introduced into a bed of particles/beads and removed therefrom by centrifuging, for example. Alternatively, particles/beads can be coated (e.g., pattern coated) onto an inert substrate (e.g., polycarbonate or polyethylene), optionally coated with 2o an adhesive, by a variety of methods (e.g., spray drying). If desired, the substrate can be microreplicated for increased surface area and enhanced clean-up. It can also be pretreated with oxygen plasma, e-beam or ultraviolet radiation, heat, or a corona treatment process.
This substrate can be used, for example, as a cover film, or laminated to a cover film, on a reservoir in a microfluidic device.
In one embodiment, the solid phase material includes a fibril matrix, which may or may not have particles enmeshed therein. The fibril matrix can include any of a wide variety of fibers. Typically, the fibers are insoluble in an aqueous environment. Examples include glass fibers, polyolefin fibers, particularly polypropylene and polyethylene microfibers, aramid fibers, a fluorinated polymer, particularly, polytetrafluoroethylene fibers, and natural cellulosic fibers. Mixtures of fibers can be used, which may be active or inactive toward binding of nucleic acid. Preferably, the fibril matrix forms a web that is at least about 15 microns, and no greater than about 1 millimeter, and more preferably, no greater than about 500 microns thick.
If used, the particles are typically insoluble in an aqueous environment. They can be made of one material or a combination of materials, such as in a coated particle. They can be swellable or nonswellable, although they are preferably nonswellable in water and organic liquids. Preferably, if the particle is doing the adhering, it is made of nonswelling, hydrophobic material. They can be chosen for their affinity for the nucleic acid. Examples of some water swellable particles are described in U.S. Pat. Nos. 4,565,663 (Errede et al.), 4,460,642 (Errede et al.), and 4,373,519 (Errede et al.). Particles that are nonswellable in to water are described in U.S. Pat. Nos. 4,810,381 (Hagen et al.), 4,906,378 (Hagen et al.), 4,971,736 (Hagen et al.); and 5,279,742 (Markell et al.). Preferred particles are polyolefin particles, such as polypropylene particles (e.g., powder). Mixtures of particles can be used, which may be active or inactive toward binding of nucleic acid.
If coated particles are used, the coating is preferably an aqueous- or organic-15 insoluble, nonswellable material. The coating may or may not be one to which nucleic acid will adhere. Thus, the base particle that is coated can be inorganic or organic. The base particles can include inorganic oxides such as silica, alumina, titanic, zirconia, etc., to which are covalently bonded organic groups. For example, covalently bonded organic groups such as aliphatic groups of varying chain length (C2, C4, C8, or C18 groups) can 2o be used.
Examples of suitable solid phase materials that include a fibril matrix are described in U.S. Pat. Nos. 5,279,742 (Markell et al.), 4,906,378 (Hagen et al.), 4,153,661 (Ree et al.), 5,071,610 (Hagen et al.), 5,147,539 (Hagen et al.), 5,207,915 (Hagen et al.), and 5,238,621 (Hagen et al.). Such materials are commercially available from 3M
Company 25 (St. Paul, MN) under the trade designations SDB-RPS (Styrene-Divinyl Benzene Reverse Phase Sulfonate, 3M Part No. 2241), cation-SR membrane (3M Part No. 2251), C-8 membrane (3M Part No. 2214), and anion-SR membrane (3M Part No. 2252).
Those that include a polytetrafluoroethylene matrix (PTFE) are particularly preferred. For example, U.S. Pat. No. 4,810,381 (Hagen et al.) discloses a solid phase 3o material that includes: a polytetrafluoroethylene fibril matrix, and nonswellable sorptive particles enmeshed in the matrix, wherein the ratio of nonswellable sorptive particles to polytetrafluoroethylene being in the range of 19:1 to 4:1 by weight, and further wherein the composite solid phase material has a net surface energy in the range of 20 to milliNewtons per meter. U.S. Pat. No. RE 36,811 (Markell et al.) discloses a solid phase extraction medium that includes: a PTFE fibril matrix, and sorptive particles enmeshed in the matrix, wherein the particles include more than 30 and up to 100 weight percent of porous organic particles, and less than 70 to 0 weight percent of porous (organic-coated or uncoated) inorganic particles, the ratio of sorptive particles to PTFE being in the range of 40:1 to 1:4 by weight.
Particularly preferred solid phase materials are available under the trade to designation EMPORE from the 3M Company, St. Paul, MN. The fundamental basis of the EMPORE technology is the ability to create a particle-loaded membrane, or disk, using any sorbent particle. The particles are tightly held together within an inert matrix of polytetrafluoroethylene (90% sorbent: 10% PTFE, by weight). The PTFE fibrils do not interfere with the activity of the particles in any way. The EMPORE membrane i5 fabrication process results in a denser, more uniform extraction medium than can be achieved in a traditional Solid Phase Extraction (SPE) column or cartridge prepared with the same size particles.
In another preferred embodiment, the solid phase (e.g., a microporous thermoplastic polymeric support) has a microporous structure characterized by a 2o multiplicity of spaced, randomly dispersed, nonuniform shaped, equiaxed particles of thermoplastic polymer connected by fibrils. Particles are spaced from one another to provide a network of micropores therebetween. Particles are connected to each other by fibrils, which radiate from each particle to the adjacent particles. Either, or both, the particles or fibrils may be hydrophobic. Examples of preferred such materials have a high 25 surface area, often as high as 40 meters2/gram as measured by Hg surface area techniques and pore sizes up to about S microns.
This type of fibrous material can be made by a preferred technique that involves the use of induced phase separation. This involves melt blending a thermoplastic polymer with an immiscible liquid at a temperature sufficient to form a homogeneous mixture, 30 forming an article from the solution into the desired shape, cooling the shaped article so as to induce phase separation of the liquid and the polymer, and to ultimately solidify the polymer and remove a substantial portion of the liquid leaving a microporous polymer matrix. This method and the preferred materials are described in detail in U.S. Patent Nos.
4,726,989 (Mrozinski), 4,957,943 (McAllister et al.), and 4,539,256 (Shipman).
Such materials are referred to as thermally induced phase separation membranes (TIPS
membranes) and are particularly preferred.
Other suitable solid phase materials include nonwoven materials as disclosed in U.S. Pat. No. 5,328,758 (Markell et al.). This material includes a compressed or fused particulate-containing nonwoven web (preferably blown microfibrous) that includes high sorptive-efficiency chromatographic grade particles.
Other suitable solid phase materials include those known as HIDE Foams, which are described, for example, in U.S. Pat. Publication No. 2003/0011092 (Tan et al.).
"HIDE" or "high internal phase emulsion" means an emulsion that includes a continuous reactive phase, typically an oil phase, and a discontinuous or co-continuous phase immiscible with the oil phase, typically a water phase, wherein the immiscible phase includes at least 74 volume percent of the emulsion. Many polymeric foams made from HIPE's are typically relatively open-celled. This means that most or all of the cells are in unobstructed communication with adjoining cells. The cells in such substantially open-celled foam structures have intercellular windows that are typically large enough to permit fluid transfer from one cell to another within the foam structure.
2o The solid phase material can include capture sites for inhibitors. Herein, "capture sites" refer to groups that are either covalently attached (e.g., functional groups) or molecules that are noncovalently (e.g., hydrophobically) attached to the solid phase material.
Preferably, the solid phase material includes functional groups that capture the inhibitors. For example, the solid phase material may include chelating groups. In this context, "chelating groups" are those that are polydentate and capable of forming a chelation complex with a metal atom or ion (although the inhibitors may or may not be .
retained on the solid phase material through a chelation mechanism). The incorporation of chelating groups can be accomplished through a variety of techniques. For example, a nonwoven material can hold beads functionalized with chelating groups.
Alternatively, the fibers of the nonwoven material can be directly functionalized with chelating groups.
Examples of chelating groups include, for example, -(CHZ-C(O)OH)2 , tris(2-aminoethyl)amine groups, iminodiacetic acid groups, nitrilotriacetic acid groups. The chelating groups can be incorporated into a solid phase material through a variety of techniques. They can be incorporated in by chemically synthesizing the material.
Alternatively, a polymer containing the desired chelating groups can be coated (e.g:, pattern coated) on an inert substrate (e.g., polycarbonate or polyethylene).
If desired, the substrate can be microreplicated for increased surface area and enhanced clean-up. It can also be pretreated with oxygen plasma, e-beam or ultraviolet radiation, heat, or a corona treatment process. This substrate can be used, for example, as a cover film, or laminated 1o to a cover film, on a reservoir in a microfluidic device.
Chelating solid phase materials are commercially available and could be used as the solid phase material in the present invention. For example, for certain embodiments of the present invention, EMPORE membranes that include chelating groups such as iminodiacetic acid (in the form of the sodium salt) are preferred. Examples of such membranes are disclosed in U.S. Pat. No. 5,147,539 (Hagen et al.) and commercially available as EMPORE Extraction Disks (47 mm, No. 2271 or 90 mm, No. 2371) from the 3M Company. For certain embodiments of the present invention, ammonium-derivatized EMPORE membranes that include chelating groups are preferred. To put the disk in the ammonium form, it can be washed with 50 mL of O.1M ammonium acetate buffer at pH
5.3 followed with several reagent water washes.
Examples of other chelating materials include, but are not limited to, crosslinked polystyrene beads available under the trade designation CHELEX from Bio-Rad Laboratories, Inc. (Hercules, CA), crosslinked agarose beads with tris(2-aminoethyl)amine, iminodiacetic acid, nitrilotriacetic acid,polyamines and polyimines as well as the chelating ion exchange resins commercially available under the trade designation DUOLITE
and DUOLITE GT73 from Rohm and Haas (Philadelphia, PA), AMBERLITE IRC-748, DIAION CR11, DUOLITE C647.
CHELEX 100 chelating resin, a styrene divinyl benzene copolymer containing iminodiacetate groups (-N-(CHZ-C(O)OH)2), has a high affinity for polyvalent metal ions 3o and can be used in certain methods of the present invention, although it is less desirable in methods carned out in microfluidic devices.
Typically, a desired concentration density of chelating groups on the solid phase material is about 0.02 nanomole per millimeter squared, although it is believed that a wider range of concentration densities is possible.
Other types of capture materials include anion exchange materials, cation exchange materials, activated carbon, reverse phase, normal phase, styrene-divinyl benzene, alumina, silica, zirconia, metal colloids. Examples of suitable anion exchange materials include strong anion exchangers such as quaternary ammonium, dimethylethanolamine, quaternary alkylamine, trimethylbenzyl ammonium, and dimethylethanolbenzyl ammonium usually in the chloride form, and weak anion exchangers such as polyamine.
Examples of suitable cation exchange materials include strong cation exchangers such as sulfonic acid typically in the sodium form, and weak cation exchangers such as carboxylic acid typically in the hydrogen form. Examples of suitable carbon-based materials include EMPORE carbon materials, carbon beads, Examples of suitable reverse phase C8 and C18 materials include silica beads that are end-capped with octadecyl groups or octyl is groups and EMPORE materials that have C8 and C18 silica beads (EMPORE
materials are available from 3M Co., St. Paul, MN). Examples of normal phase materials include hydroxy groups and dihydroxy groups.
Commercially available materials can also be modified or directly used in methods of the present invention, particularly in microfluidic devices. For example, solid phase 2o materials available under the trade designation LYSE AND GO (Pierce, Rockford, IL), RELEASE-IT (CPG, NJ), GENE FIZZ (Eurobio, France), GENE RELEASER
(Bioventures Inc., Murfreesboro, TN), and BUGS N BEADS (GenPoint, Oslo, Norway), as well as Zymo's beads (Zymo Research, Orange, CA) and Dynal's beads (Dynal, Oslo, Norway) can be incorporated into the methods of the present invention, particularly into a 25 microfluidic device as the solid phase capture material.
In certain embodiments of such methods, the solid phase material includes a coating reagent. The coating reagent is preferably selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric burner, and combinations thereof. In certain embodiments of such methods, the solid phase material 30 includes a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material, wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric burner, and combinations thereof.
Examples of suitable surfactants are listed below.
Examples of suitable strong bases include NaOH, KOH, LiOH, NH40H, as well as primary, secondary, or tertiary amines.
Examples of suitable polyelectrolytes include, polystryene sulfonic acid (e.g., poly(sodium 4-styrenesulfonate) or PSSA), polyvinyl phosphonic acid, polyvinyl boric acid, polyvinyl sulfonic acid, polyvinyl sulfuric acid, polystyrene phosphonic acid, polyacrylic acid, polymethacrylic acid, lignosulfonate, carrageenan, heparin, chondritin l0 sulfate, and salts or other derivatives thereof.
Examples of suitable selectively permeable polymeric barriers include polymers such as acrylates, acryl amides, azlactones, polyvinyl alcohol, polyethylene imine, polysaccharides. Such polymers can be in a variety of forms. They can be water-soluble, water-swellable, water-insoluble, hydrogels, etc. For example, a polymeric barrier can be prepared such that it acts as a filter for larger particles such as white blood cells, nuclei, viruses, bacteria, as well as nucleic acids such as human genomic DNA and proteins These surfaces could be tailored by one of skill in the art to separate on the basis of size and/or charge by appropriate selection of functional groups, by cross-linking, and the like.
Such materials would be readily available or prepared by one of skill in the art.
2o Preferably, the solid phase material is coated with a surfactant without washing any surfactant excess away, although the other coating reagents can be rinsed away if desired.
Typically, the coating can be carried out using a variety of methods such as dipping, rolling, spraying, etc. The coating reagent-loaded solid phase material is then typically dried, for example, in air, prior to use.
Particularly desirable are solid phase materials that are coated with a surfactant, preferably a nonionic surfactant. This can be accomplished according to the procedure set forth in the Examples Section. Although not intending to be limited by theory, the addition of the surfactant is believed to increase the wettability of the solid phase material, which allows the inhibitors to soak into the solid phase material and bind thereto.
3o The coating reagent for the solid phase materials are preferably aqueous-based solutions, although organic solvents (alcohols, etc.) can be used, if desired.
The coating reagent loading should be sufficiently high such that the sample is able to wet out the solid phase material. It should not be so high, however, that there is significant elution of the coating reagent itself. Preferably, if the coating reagent is eluted with the nucleic acid, there is no more than about 2 wt-% coating reagent in the eluted sample.
Typically, the coating solution concentrations can be as low as 0.1 wt-% coating reagent in the solution and as high as 10 wt-% coating reagent in the solution.
SURFACTANTS
Nonionic Surfactants. A wide variety of suitable nonionic surfactants are known that can be used as a lysing reagent (discussed above), an eluting reagent (discussed below), and/or as a coating on the solid phase material. They include, for example, polyoxyethylene surfactants, carboxylic ester surfactants, carboxylic amide surfactants, etc.
Commercially available nonionic surfactants include, n-dodecanoylsucrose, n-dodecyl-(3-D-glucopyranoside, n-octyl-(3-D-maltopyranoside, n-octyl-~3-D-thioglucopyranoside, n-decanoylsucrose, n-decyl-(3-D-maltopyranoside, n-decyl-(3-D-thiomaltoside, n-heptyl-~3-D-glucopyranoside, n-heptyl-~3-D-thioglucopyranoside, n-hexyl-~3-D-glucopyranoside, n-nonyl-~3-D-glucopyranoside, n-octanoylsucrose, n-octyl-~3-D-glucopyranoside, cyclohexyl-n-hexyl-(3-D-maltoside, cyclohexyl-n-methyl-(3-D-maltoside, digitonin, and those available under the trade designations PLURONIC, TRTTON, TWEEN, as well as numerous others commercially available and listed in the Kirk Othmer Technical Encyclopedia.
Examples are listed in Table 1 below. Preferred surfactants are the polyoxyethylene surfactants.
More preferred surfactants include octyl phenoxy polyethoxyethanol.
Table 1 SURFACTANT
TRADE NAME NONIONIC SURFACTANT SUPPLIER
PLURONIC F127 Modified oxyethylated alcohol Sigma and/or ox ro lated strai ht chain St. Louis, alcohols MO
TWEEN 20 Polyoxyethylene (20) sorbitan Sigma monolaurate St. Louis, MO
TRITON X-100 t-Octyl phenoxy polyethoxyethanolSigma St. Louis, MO
BRIJ 97 ~ Polyoxyethylene (10) oleyl Sigma ether ~
St. Louis, MO
IGEPAL CA-630 Octyl phenoxy poly (ethyleneoxy)Sigma ethanol St. Louis, MO
TOMADOL 1-7 Ethoxylated alcohol Tomah Products Milton, WI
Vitamin E TPGSd-Alpha tocopheryl polyethyleneEastman glycol 1000 Kingsport, TN
Also suitable are fluorinated nonionic surfactants of the type disclosed in U.S. Pat.
Publication Nos. 2003/0139550 (Savu et al.) and 2003/0139549 (Savu et al.).
Other nonionic fluorinated surfactants include those available under the trade designation ZONYL from DuPont (Wilmington, DE).
Zwitterionic Surfactants. A wide variety of suitable zwitterionic surfactants are known that can be used as a coating on the solid phase material, as a lysing reagent, and/or as an eluting reagent. They include, for example, alkylamido betaines and amine oxides thereof, alkyl betaines and amine oxides thereof, sulfo betaines, hydroxy sulfo betaines, amphoglycinates, amphopropionates, balanced amphopolycarboxyglycinates, and alkyl polyaminoglycinates. Proteins have the ability of being charged or uncharged depending on the pH; thus, at the right pH, a protein, preferably with a pI of about 8 to 9, such as modified Bovine Serum Albumin or chymotrypsinogen, could function as a zwitterionic surfactant. A specific example of a zwitterionic surfactant is cholamido propyl dimethyl ammonium propanesulfonate available under the trade designation CHAPS from Sigma.
More preferred surfactants include N-dodecyl-N,N dimethyl- 3- ammonia-1-propane sulfonate.
Cationic Surfactants. A wide variety of suitable cationic surfactants are known that can be used as a lysing reagent, an eluting reagent, andlor as a coating on the solid phase material. They include, for example, quaternary ammonium salts, polyoxyethylene alkylamines, and alkylamine oxides. Typically, suitable quaternary ammonium salts include at least one higher molecular weight group and two or three lower molecular weight groups are linked to a common nitrogen atom to produce a cation, and wherein the electrically-balancing anion is selected from the group consisting of a halide (bromide, chloride, etc.), acetate, nitrite, and lower alkosulfate (methosulfate, etc.).
The higher molecular weight substituent(s) on the nitrogen is/are often (a) higher alkyl group(s), containing about 10 to about 20 carbon atoms, and the lower molecular weight substituents may be lower alkyl of about 1 to about 4 carbon atoms, such as methyl or ethyl, which may be substituted, as with hydroxy, in some instances. One or more of the substituents may include an aryl moiety or may be replaced by an aryl, such as benzyl or phenyl. Among the possible lower molecular weight substituents are also lower alkyls of about 1 to about 4 carbon atoms, such as methyl and ethyl, substituted by lower polyalkoxy moieties such as polyoxyethylene moieties, bearing a hydroxyl end group, and falling within the general formula:
R(CHZCH20)~"_I~CH2CH 20H
where R is a (C1-C4)divalent alkyl group bonded to the nitrogen, and n represents an integer of about 1 to about 15. Alternatively, one or two of such lower polyalkoxy moieties having terminal hydroxyls may be directly bonded to the quaternary nitrogen instead of being bonded to it through the previously mentioned lower alkyl.
Examples of useful quaternary ammonium halide surfactants for use in the present invention include but are not limited to methyl- bis(2-hydroxyethyl)coco-ammonium chloride or oleyl-ammonium chloride, (ETHOQUAD C/12 and O/12, respectively) and methyl polyoxyethylene (15) octadecyl ammonium chloride (ETHOQUAD 18/25) from Akzo 2o ChemicalInc.
Anionic Surfactants. A wide variety of suitable anionic surfactants are known that can be used as a lysing reagent, an eluting reagent, and/or as a coating on the solid phase material. Surfactants of the anionic type that are useful include sulfonates and sulfates, such as alkyl sulfates, alkylether sulfates, alkyl sulfonates, alkylether sulfonates, alkylbenzene sufonates, alkylbenzene ether sulfates, alkylsulfoacetates, secondary alkane sulfonates, secondary alkylsulfates and the like. Many of these can include polyalkoxylate groups (e.g., ethylene oxide groups and/or propylene oxide groups, which can be in a random, sequential, or block arrangement) and/or cationic counterions such as Na, K, Li, 3o ammonium, a protonated tertiary amine such as triethanolamine or a quaternary ammonium group. Examples include: alkyl ether sulfonates such as lauryl ether sulfates available under the trade designation POLYSTEP B 12 and B22 from Stepan Company, Northfield, IL, and sodium methyl taurate available under the trade designation NIKKOL
CMT30 from Nikko Chemicals Co., Tokyo, Japan); secondary alkane sulfonates available under the trade designation HOSTAPUR SAS, which is a sodium (C 14-C
17)secondary alkane sulfonates (alpha-olefin sulfonates), from Clariant Corp., Charlotte, NC; methyl-2-sulfoalkyl esters such as sodium methyl-2-sulfo(C 12-C 16)ester and disodium 2-sulfo(C 12-C16)fatty acid available from Stepan Company under the trade designation ALPHASTE
PC-48; alkylsulfoacetates and alkylsulfosuccinates available as sodium laurylsulfoacetate (trade designation LANTHANOL LAL) and disodiumlaurethsulfosuccinate (trade 1o designation STEPANMILD SL3), both from Stepan Co.; and alkylsulfates such as ammoniumlauryl sulfate commercially available under the trade designation STEPANOL
AM from Stepan Co.
Another class of useful anionic surfactants include phosphates such as alkyl phosphates, alkylether phosphates, aralkylphosphates, and aralkylether phosphates. Many of these can include polyalkoxylate groups (e.g., ethylene oxide groups and/or propylene oxide groups, which can be in a random, sequential, or block arrangement).
Examples include a mixture of mono-, di- and tri-(alkyltetraglycolether)-o-phosphoric acid esters generally referred to as trilaureth-4-phosphate commercially available under the trade designation HOSTAPHAT 340KL from Clariant Corp., and PPG-5 ceteth 10 phosphate 2o available under the trade designation CRODAPHOS SG from Croda Inc., Parsipanny, NJ, as well as alkyl and alkylamidoalkyldialkylamine oxides. Examples of amine oxide surfactants include those commercially available under the trade designations AMMONYX LO, LMDO, and CO, which are lauryldimethylamine oxide, laurylamidopropyldimethylamine oxide, and cetyl amine oxide, all from Stepan Co.
ELUTION TECHNIQUES
For embodiments that use a solid phase material for retaining inhibitors, the more concentrated region of the sample that includes nucleic acid-containing material (e.g., nuclei) and/or released nucleic acid can be eluted using a variety of eluting reagents. Such 3o eluting reagents can include water (preferably RNAse-free sterile water), a buffer, a surfactant, which can be cationic, anionic, nonionic, or zwitterionic, or a strong base.
Preferably the eluting reagent is basic (i.e., greater than 7). For certain embodiments, the pH of the eluting reagent is at least 8. For certain embodiments, the pH
of the eluting reagent is up to 10. For certain embodiments, the pH of the eluting reagent is up to 13. If the eluted nucleic acid is used directly in an amplification process such as PCR, the eluting reagent should be formulated so that the concentration of the ingredients will not inhibit the enzymes (e.g., Taq Polymerise) or otherwise prevent the amplification reaction.
Examples of suitable surfactants include those listed above, particularly, those known as SDS, TRITON X-100, TWEEN, fluorinated surfactants, and PLURONICS. The l0 surfactants are typically provided in aqueous-based solutions, although organic solvents (alcohols, etc.) can be used, if desired. The concentration of a surfactant in an eluting reagent is preferably at least 0.1 weight/volume percent (w/v-%), based on the total weight of the eluting reagent. The concentration of a surfactant in an eluting reagent is preferably no greater than 1 w/v-%, based on the total weight of the eluting reagent. A
stabilizer, such as polyethylene glycol, can optionally be used with a surfactant.
Examples of suitable elution buffers include TRIS-HCI, N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), 3-[N-morpholino]propanesulfonic acid (MOPS), piperazine-N,N'-bis[2-ethanesulfonic acid]
(PIPES), 2-[N-morpholino]ethansulfonic acid (MES), TRIS-EDTA (TE) buffer, sodium citrate, ammonium acetate, carbonate salts, and bicarbonates etc.
The concentration of an elution buffer in an eluting reagent is preferably at least 10 millimolar (mM). The concentration of a surfactant in an eluting reagent is preferably no greater than 2 weight percent (wt-%).
Typically, elution of the nucleic acid-containing material and/or released nucleic acid is preferably accomplished using an alkaline solution. Although not intending to be bound by theory, it is believed that an alkaline solution allows for improved binding of inhibitors, as compared to elution with water. The alkaline solution also facilitates lysis of nucleic acid-containing material. Preferably, the alkaline solution has a pH
of 8 to 13, and more preferably 13. Examples of sources of high pH include aqueous solutions of NaOH, KOH, LiOH, quaternary nitrogen base hydroxide, tertiary, secondary or primary amines, etc. If an alkaline solution is used for elution, it is typically neutralized in a subsequent step, for example, with TRIS buffer, to form a PCR-ready sample.
The use of an alkaline solution can selectively destroy RNA, to allow for the analysis of DNA. Otherwise, RNAse can be added to the formulation to inactivate RNA, followed by heat inactivation of the RNAse. Similarly, DNAse can be added to selectively destroy DNA and allow for the analysis of RNA; however, other lysis buffers (e.g., TE) that do not destroy RNA would be used in such methods. The addition of RNAse inhibitor such as RNAsin can also be used in a formulation for an RNA preparation that is subjected to real-time PCR.
to Elution is typically carned out at room temperature, although higher temperatures may produce higher yields. For example, the temperature of the eluting reagent can be up to 95°C if desired. Elution is typically carried out within 10 minutes, although 1-3 minute elution times are preferred.
Examples of devices for using the methods of the present invention include standard laboratory filter holders furnished by companies such as Millipore, Inc. (Bedford, MA), Bio-Rad, Inc. (Hercules, CA), Osmonics, Inc. (Westborough, MA), and Whatman, Inc. (Clifton, NJ). The method of the invention can be conducted in filtration devices 2o which facilitate the movement of solutions through solid phase materials (referred to as flow-through devices) by means including centrifugation, suction, pressure.
Other devices include microtiter plates and microfluidic devices.
The present invention also provides a kit, which can include a solid phase material either with or without a holder (for example, a filter holder such as a syringe filter holder 25 or a spin filter holder, or a column with retaining frits at each end for retaining particulate material), a lysing reagent (particularly a surfactant such as a nonionic surfactant, either neat or in a solution), and instructions for binding inhibitors and eluting the nucleic acid.
Preferably, the present invention provides kits that include a flow-through receptacle (more preferably, a microfluidic device) having a solid phase (preferably, polymeric) 3o material therein, and preferably a nonionic surfactant.
Other components that could be included within kits of the present invention include conventional reagents such as wash solutions, coupling buffers, quenching buffers, blocking buffers, elution buffers, and the like. Other components that could be included within kits of the present invention include conventional equipment such as spin columns, cartridges, 96-well filter plates, syringe filters, collection units, syringes, and the like.
The kits typically include packaging material, which refers to one or more physical structures used to house the contents of the kit. The packaging material can be constructed by well-known methods, preferably to provide a sterile, contaminant-free environment.
The packaging material may have a label that indicates the contents of the kit. 1n addition, the kit contains instructions indicating how the materials within the kit are employed. As used herein, the term "package" refers to a solid matrix or material such as glass, plastic, paper, foil, and the like.
"Instructions" typically include a tangible expression describing the various methods of the present invention, including lysing conditions (e.g., lysing reagent type and concentration), the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like.
Although the methods can be used in a variety of devices, a variety of illustrative embodiments of preferred devices are described in U.S. Patent Publication Nos.
2002/0047003 (published April 25, 2003, Bedingham et al.).
2o Preferred devices useful in methods of the present invention include microfluidic devices. These typically employ a body structure that has an integrated microfluidic channel network disposed therein. In preferred aspects, the body structure of the microfluidic devices include an aggregation of two or more separate layers which, when appropriately mated or joined together, form the microfluidic device of the invention, e.g., containing the channels and/or chambers described herein. Typically, useful microfluidic devices include a top portion, a bottom portion, and an interior portion, wherein the interior portion substantially defines the channels and chambers of the device. Typically, the chambers include valves (e.g., valve septums) and are referred to as valued chambers.
A particularly preferred device for certain embodiments herein is referred to as a 3o variable valve device and is disclosed in Applicants' Assignee's copending U.S. Patent Application Serial No. 10/734,717, filed on December 12, 2003, entitled Variable Valve Apparatus and Method. In this variable valve device, the valve structures allow for removal of selected portions of the sample material located within the process chamber (i.e., the variable valued process chamber). Removal of the selected portions is achieved by forming an opening in a valve septum at a desired location.
The valve septums are preferably large enough to allow for adjustment of the location of the opening based on the characteristics of the sample material in the process chamber. If the sample processing device is rotated after the opening is formed, the selected portion of the material located closer to the axis of rotation exits the process chamber through the opening. The remainder of the sample material cannot exit through to the opening because it is located farther from the axis of rotation than the opening.
The openings in the valve septum may be formed at locations based on one or more characteristics of the sample material detected within the process chamber. It may be preferred that the process chambers include detection windows that transmit light into and/or out of the process chamber. Detected characteristics of the sample material may 15 include, e.g., the free surface of the sample material (indicative of the volume of sample material in the process chamber). Forming an opening in the valve septum at a selected distance radially outward of the free surface can provide the ability to remove a selected volume of the sample material from the process chamber.
For sample materials that can be separated into various components, e.g., whole 2o blood, rotation of the sample processing device may result in separation of the plasma and red blood cell components, thus allowing for selective removal of the components to, e.g., different process chambers.
In some embodiments, it may be possible to remove selected aliquots of the sample material by forming openings at selected locations in one or more valve septums. The 25 selected aliquot volume can be determined based on the radial distance between the openings (measured relative to the axis of rotation) and the cross-sectional area of the process chamber between the opening.
The openings in the valve septums are preferably formed in the absence of physical contact, e.g., through laser ablation, focused optical heating, etc. As a result, the openings 3o can preferably be formed without piercing the outermost layers of the sample processing device, thus limiting the possibility of leakage of the sample material from the sample processing device.
In one aspect, the present invention uses a valued process chamber in a sample processing device (e.g., a microfluidic device), the valued process (e.g., heating, mixing, lysing, combining fluids) chamber including a process chamber having a process chamber volume located between opposing first and second major sides.of the sample processing device, wherein the process chamber occupies a process chamber area in the sample processing device, and wherein the process chamber area has a length and a width transverse to the length, and further wherein the length is greater than the width. The variable valued process chamber also includes a valve chamber located within the process chamber area, the valve chamber located between the process chamber volume and the second major side of the sample processing device, wherein the valve chamber is isolated from the process chamber by a valve septum separating the valve chamber and the process chamber, and wherein a portion of the process chamber volume lies between the valve septum and a first major side of the sample processing device. A detection window is located within the process chamber area, wherein the detection window is transmissive to selected electromagnetic energy directed into and/or out of the process chamber volume.
In another aspect, the present invention provides a method that allows for the selective removal of a portion of a sample from a variable valued process chamber. The 2o method includes providing a sample processing device (e.g., a microfluidic device) as described above, providing sample material in the process chamber; detecting a characteristic of the sample material in the process chamber through the detection window;
and forming an opening in the valve septum at a selected location along the length of the process chamber, wherein the selected location is correlated to the detected characteristic of the sample material. The method also includes moving only a portion of the sample material from the process chamber into the valve chamber through the opening formed in the valve septum 3o In one embodiment, the present invention provides a method of isolating nucleic acid from a sample. In this illustrative method, the inhibitors are contained within cells, but it is understood that this may not always be the case, and methods of the present invention can be used with such samples.
The method includes: providing a sample including nucleic acid-containing material and cells containing inhibitors (such nucleic acid-containing material and cells containing inhibitors may be the same or different); contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample including nucleic acid-containing material and inhibitors; forming a concentrated region of the lysed sample; wherein the concentrated region of the lysed sample includes nucleic acid-containing material and inhibitors; substantially separating 1 o the concentrated region from a less concentrated region of the lysed sample; contacting the separated concentrated region of the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material includes a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barner, and combinations thereof;
optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nucleic acid-containing 2o material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
The nucleic acid-containing material and cells containing inhibitors may be the same or different, although they are typically different. That is, the nucleic acid containing material and the inhibitor-containing cells could potentially be the same. For example, if the sample is a huffy coat, the nucleic acid containing material can be a white blood cell, which includes both nuclei and inhibitors. If a lysing reagent (e.g., a nonionic surfactant) is used that will lyse the cell membranes of the white blood cells but not the nuclei included therein, then the inhibitors are released as are in-tact nuclei, which is also considered to be nucleic acid-containing material as defined herein.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method includes: providing a sample including cells containing inhibitors and cells containing nuclei (such cells containing inhibitors and cells containing nuclei may be the same or different); contacting the biological sample with a nonionic surfactant under conditions effective to break cell membranes and release nuclei and inhibitors and form a lysed sample; forming a concentrated region of the lysed sample;
wherein the concentrated region of the sample includes nuclei and inhibitors;
substantially separating the concentrated region of the lysed sample from the less concentrated region of the sample; contacting the separated concentrated region of the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material includes a polytetrafluoroethylene fibril matrix, to sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric burner, and combinations thereof; optionally further lysing the nuclei to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nucleic acid-containing and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
The cells containing inhibitors and cells containing nuclei may be the same or different, although they are typically different cells. That is, the nuclei-containing cells 2o and the inhibitor-containing cells could potentially be the same. For example, the inhibitors could include nuclear proteins, as well as cellular proteins.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method includes: providing a microfluidic device including a loading chamber, a valued process chamber, and a separation chamber including a solid phase material; providing a sample including nucleic acid-containing material and cells containing inhibitors (such nucleic acid-containing material and cells containing inhibitors may be the same or different); placing the sample in the loading chamber; contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample including nucleic 3o acid-containing material and inhibitors; transferring the lysed sample to a valued process chamber; forming a concentrated region of the lysed sample in the valued process chamber, wherein the concentrated region of the lysed sample includes nucleic acid-containing material and inhibitors; substantially separating the concentrated region from a less concentrated region of the lysed sample; transferring the separated concentrated region of the lysed sample to the separation chamber for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material includes capture sites (preferably chelating functional groups), a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof; optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto. As discussed above, the nucleic acid-containing material and cells containing inhibitors may be the same or different, although they are typically different.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method includes: providing a microfluidic device including a loading chamber, a valued process chamber, and a separation chamber including a solid phase material; providing a sample including cells containing inhibitors and cells containing nuclei (such cells containing inhibitors and cells containing nuclei may be the same or different); placing the sample in the loading chamber;
contacting the sample with a nonionic surfactant under conditions effective to break cell membranes and release nuclei and inhibitors to form a lysed sample; transferring the lysed sample to a valued process chamber; forming a concentrated region of the lysed sample in the valued process chamber, wherein the concentrated region of the lysed sample includes nuclei and inhibitors; substantially separating the concentrated region from a less concentrated region of the lysed sample; transferring the separated concentrated region of the lysed sample to the separation chamber for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material includes capture sites (preferably chelating functional groups), a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof; optionally further lysing the nuclei to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nucleic acid-containing and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto. As discussed above, the cells containing inhibitors and cells containing nuclei may be the same or different, although they are typically different cells.
In certain embodiments of such methods, the nucleic acid-containing material l0 includes nuclei and the method includes separating at least a portion of the nuclei from the solid phase material. That is, in-tact nuclei contact the solid phase material.
In certain embodiments of such methods, separating at least a portion of the nucleic acid-containing material (e.g., nuclei) and/or nucleic acid from the solid phase material includes transferring the nucleic acid-containing material (e.g., nuclei) and/or nucleic acid to an amplification reaction chamber (e.g., PCR sample chamber).
In certain embodiments of such methods, the coating reagent on the solid phase material includes a surfactant. Preferably, the surfactant is a nonionic or a zwitterionic surfactant, and more preferably, a nonionic surfactant.
In certain embodiments of such methods, separating at least a portion of the nucleic acid-containing material (e.g., nuclei) and/or nucleic acid includes eluting at least a portion of the nucleic acid-containing material (e.g., nuclei) and/or nucleic acid from the solid phase material with an eluting reagent.
In certain embodiments of such methods of the invention, if desired the methods can include further lysing the nucleic acid-containing material (e.g., nuclei) using a second lysing reagent.
Alternatively, in certain embodiments of such methods, the eluting reagent is also a lysing reagent. For elution and lysing simultaneously, a strong base, such as NaOH is preferred. For elution without lysing, water or TE buffer is preferred. For elution of a prelysed sample, any elution reagent (i.e., eluting reagent) can be used.
Refernng to Figure 1, a preferred embodiment of the microfluidic device suitable for use with these embodiments includes a loading chamber 10, an optional mixing chamber 12, a valued process chamber 14, a separation chamber 16 that includes a solid phase material, an eluting reagent chamber 18, a waste chamber 20 and an optional amplification reaction chamber 22. A microfluidic device that includes such a valued process chamber is disclosed, for example, in Applicants' Assignee's copending U.S.
Patent Application Serial No. 10/734,717, filed on December 12, 2003, entitled Variable Valve Apparatus and Method. These chambers are in fluid communication with each other such that a sample can be loaded into the loading chamber 10, which can then be transferred to the mixing chamber 12, or if it is not present, directly to the valued process chamber 14. If the sample is premixed with a lysing reagent (i.e., pre-lysed), for example, to then the mixing chamber 12 may not be needed. Alternatively, the mixing chamber 12 could include a lysing reagent, for example, in a pre-deposited (and typically, a dried-down) form. The sample can be concentrated in the valued process chamber 14 by a variety of means, typically by centrifugation. The valve of the valued process chamber 14 is typically positioned such that a concentrated region of a sample (that includes target nucleic acid-containing material and/or nucleic acid and inhibitors) can be substantially separated from the less concentrated region of the sample (that can also include inhibitors and a lesser amount of the target nucleic acid-containing material and/or nucleic acid).
The less concentrated region of the sample is typically transferred to the waste chamber 20. The more concentrated region of the sample is transferred to the separation chamber 16 for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material. The eluting reagent in the eluting reagent chamber 18 is then transferred to the separation chamber 16 to remove at least a portion of the target nucleic acid-containing material and/or nucleic acid. In some cases, it may be useful to archive the nuclei in the consumable for later use. In certain embodiments, this material can be directly transferred to an amplification reaction chamber 22 for carrying out a PCR
process, for example. The amplification reaction chamber 22 can optionally include pre-deposited (and typically, dried-down) reactants for the amplification reaction (e.g., PCR).
3o In another embodiment, the present invention provides a method of isolating nucleic acid from a sample. In this illustrative method, the inhibitors are contained within cells, but it is understood that this may not always be the case, and methods of the present invention can be used with such samples.
The method includes: providing a sample including nucleic acid-containing material and cells containing inhibitors (which may be the same or different);
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample including nucleic acid-containing material and inhibitors; contacting the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material includes a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the lysed sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic surfactant.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method includes: providing a sample including nucleic acid-containing material and cells containing inhibitors (which may be the same or different); contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample. including nucleic acid-containing material and inhibitors; contacting the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material includes a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barner, and 3o combinations thereof; separating at least a portion of the nucleic acid-containing material from the solid phase material having at least a portion of the inhibitors adhered thereto;
and after separating the nucleic acid-containing material, further lysing the nucleic acid-containing material to release nucleic acid.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method includes: providing a microfluidic device including a loading chamber and a separation chamber including a solid phase material;
providing a sample including nucleic acid-containing material and cells containing inhibitors (which may be the same or different); placing the sample in the loading chamber; contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample including nucleic acid-containing material and inhibitors; transferring the lysed sample to the separation chamber to contact the solid phase material and to preferentially adhere at least a portion of the inhibitors to the solid phase material; optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the sample with the solid phase material; wherein the solid phase material includes capture sites (e.g., chelating functional groups), a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barner, and combinations thereof; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic surfactant.
In another embodiment, the present invention provides a method of isolating nucleic acid from a sample, the method includes: providing a microfluidic device including a loading chamber and a separation chamber including a solid phase material;
providing a sample including nucleic acid-containing material and cells containing inhibitors (which may be the same or different); placing the sample in the loading chamber; contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample including nucleic acid-containing material and inhibitors; transferring the lysed sample to the separation 3o chamber to contact the solid phase material and to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material includes capture sites (e.g., chelating functional groups), a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barner, and combinations thereof; and separating at least a portion of the nucleic acid-containing material from the solid phase material having at least a portion of the inhibitors adhered thereto; and after separating the nucleic acid-containing material, further lysing the nucleic acid-containing material to release nucleic acid.
As discussed above with respect to Illustrative Method l, the nucleic acid-containing material and cells containing inhibitors of the embodiments of Illustrative 1o Method 2 may be the same or different, although they are typically different. That is, the nucleic acid containing material and the inhibitor-containing cells could potentially be the same. Also, in illustrative Method 2, the sample can include free nucleic acid and free inhibitors.
In certain embodiments of such methods, separating the nucleic acid-containing material (e.g., nuclei) (and/or nucleic acid if already isolated from the nucleic acid-containing material) from the solid phase material includes transferring the nucleic acid-containing material (e.g., nuclei) (and/or nucleic acid) to an amplification reaction chamber (e.g., a PCR sample chamber).
In certain embodiments of such methods, the solid phase material includes a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material, wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof.
1n certain embodiments of such methods, separating includes eluting at least a portion of the nucleic acid-containing material (e.g., nuclei) (and/or nucleic acid) from the solid phase material with an eluting reagent.
Refernng to Figure 2, a preferred embodiment of the microfluidic device suitable for use with these embodiments includes a loading chamber 30, an optional mixing chamber 32, a separation chamber 36 that includes a solid phase material, an eluting reagent chamber 38, and an optional amplification chamber 42. These chambers are in fluid communication with each other such that a sample can be loaded into the loading chamber 30, which can then be transferred to the mixing chamber 32, or if it is not present, directly to the separation chamber 36. If the sample is premixed with a lysing reagent (i.e., pre-lysed), for example, then the mixing chamber 32 may not be needed.
Alternatively, the mixing chamber 32 could include a lysing reagent, for example, in a pre-deposited (and typically, a dried-down) form. The lysed sample is transferred to the separation chamber 36 for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material. The eluting reagent in the eluting reagent chamber 38 is then transferred to the separation chamber 36 to remove at least a portion of the target nucleic acid-containing material and/or nucleic acid. The sample can be eluted with io a strong base for further lysing. After lysis using a strong base, in some cases, the samples can be heated for a few minutes at high temperatures (95°C) to denature proteins that are inhibitory to PCR followed by neutralization using an agent such as TRIS
buffer to adjust the pH of the sample prior to PCR. Alternatively, the eluted sample can be transferred to a chamber for further lysing using other methods (e.g., heating, Proteinase K).
Alternatively, these reagents could be pre-deposited (and dried-down) if desired. In certain embodiments, this material can be directly transferred to an amplification reaction chamber 42 for carrying out a PCR process, for example. The amplification reaction chamber 42 can optionally include pre-deposited reactants for the amplification reaction (e.g., PCR).
ADDITIONAL EMBODIMENTS
In certain embodiments of such methods that involve the use of a microfluidic device, placing the sample in the loading chamber occurs prior to contacting the sample with a first lysing reagent. Alternatively, placing the sample in the loading chamber occurs after contacting the sample with a first lysing reagent. The first lysing reagent can be water or a nonionic surfactant, for example.
If additional lysing is needed to release nucleic acid from nucleic acid-containing material (e.g., nuclei), other lysing conditions can be used. For example, this includes subjecting the nucleic acid-containing material to a strong base with optional heating. The 3o strong base is typically NaOH, but can be others such as KOH, LiOH, NH40H, as well as primary, secondary, or tertiary amines. Typically, the temperature is at room temperature.
If a base is used, the sample containing the released nucleic acid may need to have its pH
adjusted, particularly if the nucleic acid is to be subjected to a subsequent amplification process. Thus, certain embodiments of the invention include adjusting the pH
of the sample typically to at least 7.5, and typically to no greater than 9.
In certain embodiments of such methods that involve the use of a microfluidic device, the first lysing reagent is a nonionic surfactant. In certain embodiments of such methods, the loading chamber includes the first lysing reagent (e.g., pre-deposited (and typically, dried-down) nonionic surfactant) and contacting the sample with a first lysing reagent occurs upon placing the sample in the loading chamber.
1o Alternatively, the sample could be transferred to a subsequent processing chamber with the first lysing reagent (preferably, a nonionic surfactant) therein. For example, contacting the sample with a first lysing reagent (preferably, a nonionic surfactant) occurs in a mixing chamber with sufficient mixing to break cell membranes and release nuclei and inhibitors to form a lysed sample. It should be understood that if a "first" lysing 15 reagent is used, the methods do not necessarily require the use of a "second" lysing reagent; rather, the term "first lysing reagent" is used herein to distinguish from any additional lysing reagents if used.
As stated above, the addition of sucrose in a buffer (particularly, a TRIS
buffer) may help in the isolation of nuclei. The buffer could also include magnesium salts and 2o surfactants such as TRITON X-100. This may also provide a good medium for lysis of white blood cells. Furthermore, in certain cases, when the nuclei need to be archived, particularly within a microfluidic device, using a nuclei storage buffer may be useful. The nuclei storage buffer could include sucrose, magnesium salts, EDTA, dithiothrietol, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), and/or glycerol, for example, in a buffer 25 (e.g., TRIS buffer) and would allow for stable storage of nuclei.
In certain embodiments of such methods that involve the use of a microfluidic device, forming a concentrated region of the sample in the valued process chamber includes centrifuging the sample in the process chamber. Typically, separating the concentrated region from the less concentrated of the sample includes transferring the less 3o concentrated region of the sample through the valve to a waste bin.
Spinning speeds, such as 400 rcf for 2 minutes, are typically desirable when using a microfluidic device, although higher spinning speeds andlor longer spinning times may be used.
The nonionic surfactant, such as TRITON X-100, can be pre-deposited (and dried down uniformly if desired) in a mixing chamber of a microfluidic device, for example, such that lysing occurs when the sample (e.g., blood) is mixed with the surfactant for a few minutes. In other cases, a dilute solution of surfactant can be pre-mixed with the sample (e.g., blood) prior to introduction into the mixing chamber.
In other cases, when a nonionic surfactant is not used, water or ammonium chloride can be used for lysis of red blood cells. After lysis occurs in the mixing chamber, the white blood cells can be centrifuged down at relatively low speeds.
In certain embodiments of the methods described herein, the sample can be whole blood. The whole blood is then typically separated into component parts and the portion containing white blood cells (typically referred to as the huffy coat) separated and lysed to release the nuclei and/or nucleic acid. For example, in certain embodiments, the method can include centrifuging the whole blood (e.g., in a valued process chamber) to form a plasma layer (often in the upper layer), a red blood cell layer (often in the lower layer), and an interfacial layer that includes white blood cells, and removing a substantial portion of the interfacial layer (i.e., huffy coat). The huffy coat can then be subjected to further processing.
In certain embodiments, the huffy coat could be separated from whole blood using conventional techniques. The huffy coat could then be used as the sample in the methods described herein.
In addition to the solid phase material discussed herein, other types of solid phase materials, particularly beads, can be introduced into a microfluidic device in a variety of embodiments of the present invention. For example, beads can be functionalized with the appropriate groups to isolate specific cells, viruses, bacteria, proteins, nucleic acids, etc.
The beads can be segregated from the sample by centrifugation and subsequent separation.
The beads could be designed to have the appropriate density and sizes (nanometers to microns) for segregation. For example, in the case of viral capture, beads that recognize the protein coat of a virus can be used to capture and concentrate the virus prior to or after removal of small amounts of residual inhibitors from a serum sample.
The inhibitors can be removed using solid phase materials, as described herein, prior to or after capture of viral particles onto the beads. In addition to this, the amount of inhibitors can be reduced using concentration/separation/optional dilution steps, for example, as disclosed in U.S. Patent Application Serial No. , filed on , entitled METHODS FOR NUCLEIC ACID ISOLATION AND KITS USING
A MICROFLUIDIC DEVICE AND CONCENTRATION STEP (Attorney Docket No.
59801US002). Such concentration/separation/optional dilution steps can be used with various methods and samples described herein.
Nucleic acids can be extracted out of the segregated viral particles by lysis.
Thus, the beads could provide a way of concentrating relevant material in a specific region within a microfluidic device, also allowing for washing of irrelevant materials and elution of relevant material from the captured particle.
Examples of such beads include, but are not limited to, crosslinked polystyrene beads available under the trade designation CHELEX from Bio-Rad Laboratories, Inc.
(Hercules, CA), crosslinked agarose beads with tris(2-aminoethyl)amine, iminodiacetic acid, nitrilotriacetic acid, polyamines and polyimines as well as the chelating ion exchange resins commercially available under the trade designation DUOLITE C-467 and DUOLITE GT73 from Rohm and Haas (Philadelphia, PA), AMBERLITE IRC-748, DIAION CR11, DUOLITE C647. These beads are also suitable for use as the solid phase 2o material as discussed above.
Other examples of beads include those available under the trade designations GENE FIZZ (Eurobio, France), GENE RELEASER (Bioventures Inc., Murfreesboro, TN), and BUGS N BEADS (GenPoint, Oslo, Norway), as well as Zymo's beads (Zymo Research, Orange, CA) and DYNAL beads (Dynal, Oslo, Norway).
Other materials are also available for pathogen capture (e.g., viral particles, bacteria). For example, polymer coatings can also be used to isolate specific cells, viruses, bacteria, proteins, nucleic acids, etc. in certain embodiments of the invention. These polymer coatings could directly be spray jetted, for example, onto the cover film of a microfluidic device.
3o Viral particles can be captured onto beads by covalently attaching antibodies onto bead surfaces. The antibodies can be raised against the viral coat proteins.
For example, DYNAL beads can be used to covalently link antibodies. Alternatively, synthetic polymers, for example, anion-exchange polymers, can be used to concentrate viral particles. Commercially available resins such as viraffinity (Biotech Support Group, East Brunswick, NJ) can be used to coat beads or applied as polymer coatings onto select locations in microfluidic device to concentrate viral particles. BUGS N BEADS
(GenPoint, Oslo, Norway) can, for example, be used for extraction of bacteria.
Here, these beads can be used to capture bacteria such as Staphylococcus, Streptococcus, E
coli, Salmonella, and Clamydia elementary bodies.
Thus, in one embodiment of the present invention when the sample includes viral l0 particles or other pathogens (e.g., bacteria), a microfluidic device can include solid phase material in the form of viral capture beads or other pathogen capture material. More specifically, in one case, the beads can be used only for concentration of virus or bacteria, for example, followed by segregation of beads to another chamber, ending with lysis of virus or bacteria. In another case, the beads can be used for concentration of virus or 15 bacteria, followed by lysis and capture of nucleic acids onto the same bead, dilution of beads, concentration of beads, segregation of beads, and repeating the process multiple times prior to elution of captured nucleic acid.
If the downstream application of the nucleic acid is subjecting it to an amplification process such as PCR, then all reagents used in the method are preferably compatible with 20 such process (e.g., PCR compatible). Furthermore, the addition of PCR
facilitators may be useful, especially for diagnostic purposes. Also, heating of the material to be amplified prior to amplification can be beneficial.
In embodiments in which the inhibitors are not completely removed, the use of buffers, enzymes, and PCR facilitators can be added that help in the amplification process 25 in the presence of inhibitors. For example, enzymes other than Taq Polymerase, such as rTth, that are more resistant to inhibitors can be used, thereby providing a huge benefit for PCR amplification. The addition of Bovine Serum Albumin, betaine, proteinase inhibitors, bovine transferrin, etc. can be used as they are known to help even further in the amplification process. Alternatively, one can use a commercially available product such as 30 Novagen's Blood Direct PCR Buffer kit (EMD Biosciences, Darmstadt, Germany) for direct amplification from whole blood without the need for extensive purification.
Objects and advantages of this invention are further illustrated by the following examples, but the particular materials and amounts thereof recited in these examples, as well as other conditions and details, should not be construed to unduly limit this invention.
EXAMPLES
Example lA: Preparation of Solid Phase Material: Ammonia Form without TRITON X-A 3M No. 2271 EMPORE Extraction Chelating Disk (3M, St. Paul, MN) was 1o placed in a glass filter holder. The extraction disk was converted into the ammonia form, following the procedure printed on the package insert.
Example 1B: Preparation of Solid Phase Material: Ammonia Form with TRITON X-A 3M No. 2271 EMPORE Extraction Chelating Disk was placed in a glass filter 15 holder. The extraction disk was converted into the ammonia form, following the procedure printed on the package insert. The disk placed in a vial and was submerged in a 1 TRITON X-100 (Sigma-Aldrich, St. Louis, MO) solution (0.1 gram (g) of TRITON X-in 10 mL of water), mixing for about 6-8 hours on a Thermolyne Vari-Mix Model Rocker (Barnstead/Thermolyne, Dubuque, IA). The disk was placed in glass filter holder, 2o dried by applying a vacuum for about 20 minutes (min), and then dried overnight at room temperature (approximately 21°C), taking care not to wash or rinse the disk.
Example 1 C: Preparation of Solid Phase Material: Anion-SR Empore Chelating Membrane with TRITON X-100 25 A 3M No. 2252 EMPORE Extraction Chelating Disk was placed in a vial and was submerged in a 1% TRITON X-100 solution (0.1 g of TRITON X-100 in 10 milliliters (mL) of water), mixing for about 6-8 hours on a Thermolyne Vari-Mix Model Rocker. The disk was placed in glass filter holder, dried by applying a vacuum for about 20 minutes, and then dried overnight at room temperature (approximately 21°C), taking 3o care not to wash or rinse the disk.
Example 1D: Preparation of Solid Phase Material: C8 Chelating Membrane with A 3M No. 2214 EMPORE Extraction Chelating Disk was placed in a vial and was submerged in a 1 % TRITON X-100 solution (0.1 g of TRITON X-100 in 10 mL of water), mixing for about 6-8 hours on a Thermolyne Vari-Mix Model M48725 Rocker. The disk was placed in glass filter holder, dried by applying a vacuum for about 20 minutes, and then dried overnight at room temperature (approximately 21°C), taking care not to wash or rinse the disk.
to Example lE: Preparation of Solid Phase Material: Chelating Membrane with TRITON X-100 and FR-2025 A 3M No. 2271 EMPORE Extraction Chelating Disk was placed in a vial and was submerged in a 50/50 mixture of 1% TRITON X-100 (0.1 g of TRITON X-100 in 10 15 milliliters (mL) of water) and 0.1% FR-2025 (3M, St. Paul, MN) and mixed for about 6-8 hours on a Thermolyne Vari-Mix Model M48725 Rocker. The disk was placed in glass filter holder, dried by applying a vacuum for about 20 minutes, and then dried overnight at room temperature (approximately 21°C), taking care not to wash or rinse the disk.
2o Example 1F: Preparation of Solid Phase Material: Chelating Membrane with TRITON X-100 and FC-4430 A 3M No. 2271 EMPORE Extraction Chelating Disk was placed in a vial and was submerged in a 50/50 mixture of 1% TRITON X-100 (0.1 g of TRITON X-100 in 10 milliliters (mL) of water) and 0.1 % FC-4430 (3M, St. Paul, MN) and mixed for about 6-8 25 hours on a Thermolyne Vari-Mix Model M48725 Rocker. The disk was placed in glass filter holder, dried by applying a vacuum for about 20 minutes, and then dried overnight at room temperature (approximately 21°C), taking care not to wash or rinse the disk.
Example 1 G: Preparation of Solid Phase Material: Chelating Membrane with TRITON
X- 100 and PSSA
A 3M No. 2271 EMPORE Extraction Chelating Disk was placed in a glass filter holder. The extraction disk was converted into the ammonia form, following the procedure printed on the package insert. The disk placed in a vial and was submerged in a 50/50 mixture of 1 % TRITON X-100 (0.1 g of TRITON X-100 in 10 milliliters (mL) of water) and 1% PSSA (Poly(sodium 4-styrene-sulfonate) (Sigma-Aldrich, St. Louis, MO) (20 wt-PSSA stock solution was diluted to 1 wt-% in RNAse-free sterile water) and mixed for about 6-8 hours on a Thermolyne Vari-Mix Model M48725 Rocker. The disk was placed l0 in glass filter holder, dried by applying a vacuum for about 20 minutes, and then dried overnight at room temperature (approximately 21°C), taking care not to wash or rinse the disk.
Example 1H: Preparation of Solid Phase Material: Cation-SR EMPORE Chelating Membrane with TRITON X-100 A 3M No. 2251 EMPORE Extraction Chelating Disk was placed in a vial and was submerged in a 1% TRITON X-100 solution (0.1 g of TRITON X-100 in 10 mL of water), mixing for about 6-8 hours on a Thermolyne Vari-Mix Model M48725 Rocker. The disk was placed in glass filter holder, dried by applying a vacuum for about 20 minutes, and then dried overnight at room temperature (approximately 21 °C), taking care not to wash or rinse the disk.
Example 1J: Preparation of Solid Phase Material: EMPORE Chelating Membrane with A 3M No. 2241 EMPORE Extraction Chelating Disk was placed in a vial and was submerged in a 1 % TRITON X-100 solution (0.1 g of TRITON X-100 in 10 mL of water), mixing for about 6-8 hours on a Thermolyne Vari-Mix Model M48725 Rocker. The disk was placed in glass filter holder, dried by applying a vacuum for about 20 minutes, and then dried overnight at room temperature (approximately 21°C), taking care not to wash or rinse the disk.
Example 2: Procedure for Isolation of Genomic DNA from Whole Blood Using Chelating Solid Phase Material One (1) pL of neat TRITON X-100 was added to one hundred (100) p,L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (for approximately S seconds every 20 seconds). The solution was investigated to make sure that it was transparent before 1 o proceeding to the next step. The solution was spun in an Eppendorf Model centrifuge (Brinkmann, Westbury, NIA at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) p.L of concentrated material at the bottom of the centrifuge tube. This material was transferred to a 3M No. 2271 EMPORE
Extraction Chelating Disk, making sure that the material was evenly distributed on the surface of the disk. The material was allowed to dry on the disk for about 2-5 minutes until the intense red color became darker. Twelve microliters (12 p,L) of 0.083 molar (M) NaOH was added to the extraction disk. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 p,L might be obtained).
The color of the sample removed from the membrane varied from colorless to faint green.
If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 p,L aliquot was removed and added to 10 p,L of 40 millimolar (mM) TRIS-HCl (Sigma-Aldrich, St. Louis, MO) (pH 7.4).
Example 3: Procedure for Isolation of Genomic DNA from Whole Blood Using Ammoniated Chelating Solid Phase Material One (1) ~.L of neat TRITON X-100 was added to one hundred (100) ~,L of whole blood. The solution was incubated at room temperature (approximately 21 °C) for about 5 minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415D
centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) p,L of concentrated material at the bottom of the centrifuge tube.
This material was transferred to the extraction membrane prepared in Example lA, making sure that the material was evenly distributed on the surface of the membrane.
The material was allowed to dry on the membrane for about 2-5 minutes until the intense red color became darker. Twelve (12) ~.L of 0.083 M NaOH was added to the extraction membrane. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 pL might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A
2 p,L
l0 aliquot was removed and added to 10 ~.L of 40 mM TRIS-HCI (pH 7.4).
Example 4: Procedure for Isolation of Genomic DNA from Whole Blood Using Ammoniated Chelating Solid Phase Material with TRITON X-100 One (1) pL of neat TRITON X-100 was added to one hundred (100) wL of whole blood. The solution was incubated at_ room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415D
centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) p.L of concentrated material at the bottom of the centrifuge tube.
This material was transferred to the extraction membrane prepared in Example 1B, making sure that the material was evenly distributed on the surface of the membrane.
The material was allowed to dry on the membrane for about 2-5 minutes until the intense red color became darker. Twelve (12) p,L of 0.083 M NaOH was added to the extraction membrane. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 ~.L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute.
The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing. A 2 p.L
aliquot was removed and added to 10 ~L of 40 mM TRIS-HCl (pH 7.4).
Example 5A: Concentration of Genomic DNA by Spinning Whole Blood Prior to Treatment with Solid Phase Material: Sample Isolation From the Top of the Tube One (1) ~,L of neat TRITON X-100 was added to one hundred (100) ~,L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415D
centrifuge at 400 rcf for about 10 minutes. A two (2) ~,L aliquot was removed from the top of the centrifuge tube and was transferred to the extraction membrane prepared in Example 1B, making sure that the material was evenly distributed on the surface of the membrane.
The material was allowed to dry on the membrane for about 2-5 minutes until the intense red color became darker. Twelve (12) ~,L of 0.083 M NaOH was added to the extraction membrane The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 ~,L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A
2 ~,L
aliquot was removed and added to 10 ~,L of 40 mM TRIS-HCl (pH 7.4).
Example 5B: Concentration of Genomic DNA by Spinning Whole Blood Prior to 2o Treatment with Solid Phase Material: Sample Isolation from the Bottom of the Tube One (1) ~L of neat TRITON X-100 was added to one hundred (100) ~,L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 541 SD
centrifuge at 400 rcf for about 10 minutes. A two (2) ~,L aliquot was removed from the bottom of the centrifuge tube and was transferred to the extraction membrane prepared in Example 1B, making sure that the material was evenly distributed on the surface of the membrane. The material was allowed to dry on the membrane for about 2-S
minutes until the intense red color became darker. Twelve (12) ~,L of 0.083 M NaOH was added to the extraction membrane. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 ~L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 p.L
aliquot was removed and added to 10 pL of 40 mM TRIS-HCl (pH 7.4).
Example SC: Purification of Genomic DNA by Treatment with Solid Phase Material:
Sample Isolation from the Top of the Tube - No Concentration Step One (1) ~L of neat TRITON X-100 was added to one hundred (100) p.L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 1o minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. A two (2) p,L aliquot was removed from the top of the centrifuge tube and was transferred to the extraction membrane prepared in Example 1B, making sure that the material was evenly distributed on the surface of the membrane. The i5 material was allowed to dry on the membrane for about 2-5 minutes until the intense red color became darker. Twelve (12) ~L of 0.083 M NaOH was added to the extraction membrane. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 ~.L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was 2o foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute.
A 2 p.L
aliquot was removed and added to 10 pL of 40 mM TRIS-HCl (pH 7.4).
Example 6A: Concentration of Genomic DNA by Spinning Whole Blood Followed by QIAamp Clean-up: Sample Isolation from the Top of the Tube 25 One (1) ~L of neat TRITON X-100 was added to one hundred (100) ~L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415D
3o centrifuge at 400 rcf for about 10 minutes. A two (2) wI, aliquot was removed from the top of the centrifuge tube and was added to 198 p,L of lx Phosphate buffer saline (PBS).
Clean DNA was obtained, following "Blood and Body Fluid Spin Protocol"
described in QIAamp DNA Blood Mini Kit Handbook p. 27(Qiagen, Valencia, CA), eluting in 72 ~,L
of water.
Example 6B: Concentration of Genomic DNA by Spinning Whole Blood Followed by QIAamp Clean-up: Sample Isolation from the Bottom of the Tube One (1) p,L of neat TRITON X-100 was added to one hundred (100) p.L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about S
minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415D
centrifuge at 400 rcf for about 10 minutes. A two (2) p,L aliquot was removed from the bottom of the centrifuge tube and was added to 198 pL of 1 x Phosphate buffer saline (PBS). Clean DNA was obtained, following "Blood and Body Fluid Spin Protocol"
described in QIAamp DNA Blood Mini Kit Handbook p. 27, eluting in 72 ~,L of water.
Example 6C: Collecttion of Genomic DNA by QIAamp Clean-up: Sample Isolation from the Top of the Tube - No Centrifuging One (1) ~,L of neat TRITON X-100 was added to one hundred (100) pL of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 s minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. A two (2) ~.L aliquot was removed from the top of the centrifuge tube and was added to 198 pL of lx Phosphate buffer saline (PBS, Sigma-Aldrich, St. Louis, MO). Clean DNA was obtained, following "Blood and Body Fluid to Spin Protocol" described in QIAamp DNA Blood Mini Kit Handbook p. 27, eluting in 72 p,L of water.
Example 7A: Effect of Inhibitor/DNA on PCR: Varying Inhibitor Concentration with Fixed DNA Concentration 15 A dilution series of inhibitors were made prior to spiking with clean human genomic DNA in order to study the effect of inhibitor on PCR. To 10 ~,L of 15 nanograms per microliter (ng/p.L) human genomic DNA, 1 wL of different Mix I (neat or dilutions thereof) was added (Samples 7 - no inhibitor added, 7D - neat, 7E - 1:10, 7F -1:30, 7G -1:100, 7H - 1:300) and vortexed. Two (2) p,L aliquots of each sample were taken for 20 ~,L
20 PCR. The results are shown in Table 2.
Mix I: one hundred (100) p,L of whole blood was added to 1 pL of neat TRITON
X-100. The solution was incubated at room temperature (approximately 21°C) for about S
minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before 25 proceeding to the next step. The solution was spun in an Eppendorf Model centrifuge at 400 rcf for about 10 minutes. Approximately 80 ~L from the top of the microcentrifuge tube and designated Mix I.
Example 7B: Effect of Inhibitor/DNA on PCR: Varying DNA Concentration with Fixed Inhibitor Concentration To 10 ~.L of human genomic DNA, 1 ~,L of 1:3 diluted Mix I (described above) was added. DNA concentrations that were examined were the following: Samples ng/~,L, 7K - 7.5 ng/wL, 7L - 3.75 ng/~L, 7M - 1.5 ngh,L. Two (2) ~L aliquots of each sample were taken for 20 ~,L PCR. The results are shown in Table 2.
Example 7C: Effect of Inhibitor/DNA on PCR: DNA with No Added Inhibitor The following samples were prepared with 1 ~,L of water added to each DNA
to sample instead of inhibitor: Samples 7N - 1 S ng/~,L, 7P - 7.5 ng/~,L, 7Q -3.75 ng/~,L, 7R -1.5 ng/~,L. Two (2) ~.L aliquots of each sample were taken for 20 ~L PCR. The results are shown in Table 2.
Table 2 Sample No. Ct (duplicateSample No. Ct (duplicate samples) samples) 7 19.10 7K 29.16 19.06 30.22 7D 13.94 7L 30.47 29.50 29.96 7E 27.39 7M 28.43 26.22 26.16 7F 21.44 7N 20.05 20.66 19.80 7G 19.90 7P 20.74 19.30 20.54 7H 19.90 7Q 21.95 20.08 21.88 7J 28.45 7R 22.67 28.61 23.10 Example 8A: Recovery of Nuclei from Solid Phase Material One (1) p.L of neat TRITON X-100 was added to 2 p,L of white blood cells (PBMCs). The solution was vortexed briefly, and was spun in an Eppendorf Model 5415D centrifuge at 400 rcf for about 1 minute. A three (3) pL was placed on a chelating membrane prepared as in Example 1B. The material was allowed to dry on the membrane for about 2-5 minutes. Thirteen (13) ~.L of 0.077 M NaOH was added to the extraction membrane. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 13 p,L might be obtained). If the solution was foamy, it to was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 p,L
aliquot was removed and added to 10 ~,L of 40 mM TRIS-HCl (pH 7.4).
Example 8B: Recovery of Nuclei with No Solid Phase Material One (1) pL of neat TRITON X-100 was added to 2 pL of white blood cells (PBMCs). The solution was vortexed briefly, and was spun in an Eppendorf Model 5415D centrifuge at 400 rcf for about 1 minute. Ten (10) p,L of 0.1 M NaOH was added to the solution and incubated at room temperature (approximately 21 °C) for about 5 minutes.
A 2 p,L aliquot was removed and added to 10 ~L of 40 mM TRIS-HCl (pH 7.4).
Example 8C: Sample Spiked Afterwards with Nuclei One (1) p,L of neat TRITON X-100 was added to 2 p,L of water. The solution was vortexed briefly, and was spun in an Eppendorf Model 5415D centrifuge at 400 rcf for about 1 minute. A three (3) ~,L was placed on a chelating membrane prepared as in Example 1B. The material was allowed to dry on the membrane for about 2-5 minutes.
Thirteen (13) ~,L of 0.077 M NaOH was added to the extraction membrane. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 13 wL might be obtained). If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. Two (2) pL of white blood cells (PBMCs) was added to a 10 p,L aliquot of the solution and incubated at room temperature (approximately 21 °C) for about S minutes. A 2 pL aliquot was removed and added to 10 ~,L of 40 mM
TRIS-HCl (pH 7.4).
Example 9A: Procedure for Isolation of Genomic DNA from Whole Blood Using Solid Phase Material: Extraction from the Solid Phase Material with NaOH
One (1) p,L of neat TRITON X-100 was added to one hundred (100) ~,L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (approximately 5 seconds every 20 seconds).
The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415D centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) pL
to of concentrated material at the bottom of the centrifuge tube. This material was transferred to the extraction membrane prepared in Example 1B, making sure that the material was evenly distributed on the surface of the membrane. The material was allowed to dry on the membrane for about 2-5 minutes until the intense red color became darker. Twelve (12) p,L of 0.083 M NaOH was added to the extraction membrane.
The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 pL might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. Fifty (50) pL of lx TE buffer (Sigma-Aldrich, St. Louis, MO) was added to a 10 p.L aliquot of the solution.
Three 2o hundred (300) p.L of 40 mM TRIS-HCl (pH 7.4) was added to the solution.
Example 9B: Procedure for Isolation of Genomic DNA from Whole Blood Using Solid Phase Material: Extraction from the Solid Phase Material with Water One (1) ~L of neat TRITON X-100 was added to one hundred (100) p,L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (approximately 5 seconds every 20 seconds).
The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415D centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) p,L
of concentrated material at the bottom of the centrifuge tube. This material was transferred to the extraction membrane prepared in Example 1B, making sure that the material was evenly distributed on the surface of the membrane. The material was allowed to dry on the membrane for about 2-5 minutes until the intense red color became darker. Twelve (12) ~.L of water was added to the extraction membrane. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 ~,L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. Fifty (50) ~.L of 0.1 M NaOH was added to a ~L aliquot of the solution and incubated at room temperature (approximately 21°C) for about S minutes. Three hundred (300) ~,L of 40 mM TRIS-HCl (pH 7.4) was added to the to solution.
Example 10: Genomic DNA Isolated from Whole Blood on Ammoniated Chelating Solid Phase Material Treated with TRITON X-100 One (1) pL of 3% TRITON X-100 was added to two (2) p,L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (approximately 5 seconds every 20 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. This material was transferred to the extraction membrane prepared in Example 1 B, making sure that the material was evenly distributed on the surface of the membrane.
2o The material was allowed to dry on the membrane for about 2-5 minutes until the intense red color became darker. Thirteen (13) ~.L of 0.083 M NaOH was added to the extraction membrane. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing. A 2 ~L aliquot was removed and added to 10 ~L of 40 mM TRIS-HCl (pH
7.4).
Example 11: Procedure for Isolation of Genomic DNA from Whole Blood Using Anion-SR Chelating Solid Phase Material One (1) wL of neat TRITON X-100 was added to one hundred (100) pL of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 3o minutes, vortexing the solution intermittently (approximately 5 seconds every ZO seconds).
The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415 D centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) ~.L
of concentrated material at the bottom of the centrifuge tube. This material was transferred to a 3M No. 2252 EMPORE Anion-SR Extraction Chelating Disk prepared as described in Example 1C, making sure that the material was evenly distributed on the surface of the disk. The material was allowed to dry on the disk for about 2-5 minutes until the intense red color became darker. Twelve (12) ~,L of 0.083 M NaOH was added to the extraction disk. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 p.I, might be obtained). The color of the l0 sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 p,L
aliquot was removed and added to 10 p.L of 40 mM TRIS-HCl (pH 7.4).
Example 12: Procedure for Isolation of Genomic DNA from Whole Blood Using C8 Chelating Solid Phase Material One (1) ~L of neat TRITON X-100 was added to one hundred (100) p,L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (approximately 5 seconds every 20 seconds).
The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415 D centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) pL
of concentrated material at the bottom of the centrifuge tube. This material was transferred to a 3M No. 2214 EMPORE C-8 Extraction Chelating Disk prepared as described in Example 1D, making sure that the material was evenly distributed on the surface of the disk. The material was allowed to dry on the disk for about 2-5 minutes until the intense red color became darker. Twelve (12) p.L of 0.083 M NaOH was added to the extraction disk. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 ~,L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 pL
aliquot was removed and added to 10 ~,L of 40 mM TRIS-HCl (pH 7.4).
Example 13: Procedure for Isolation of Genomic DNA from Whole Blood Using Chelating Solid Phase Material One (1) wL of neat TRITON X-100 was added to one hundred (100) p,L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (approximately 5 seconds every 20 seconds).
The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415 D centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) pL
of concentrated material at the bottom of the centrifuge. tube. This material was transferred to a 3M No. 2271 EMPORE Extraction Chelating Disk prepared as described in Example lE, making sure that the material was evenly distributed on the surface of the disk. The material was allowed to dry on the disk for about 2-5 minutes until the intense red color became darker. Twelve (12) p,L of 0.083 M NaOH was added to the extraction disk. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 p,L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 ~,L aliquot was removed and added to 10 ~,L of 40 mM TRIS-HCl (pH 7.4).
Example 14: Procedure for Isolation of Genomic DNA from Whole Blood Using Chelating Solid Phase Material One (1) ~.L of neat TRITON X-100 was added to one hundred (100) p.L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (approximately 5 seconds every 20 seconds).
The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415 D centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) p,L
of concentrated material at the bottom of the centrifuge tube. This material was transferred to a 3M No. 2271 EMPORE Extraction Chelating Disk prepared as described in Example 1F, making sure that the material was evenly distributed on the surface of the disk. The material was allowed to dry on the disk for about 2-5 minutes until the intense red color became darker. Twelve (12) p.L of 0.083 M NaOH was added to the extraction disk. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 ~,L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 ~.L aliquot was removed and added to 10 p,L of 40 mM TRIS-HCl (pH 7.4).
Example 15: Procedure for Isolation of Genomic DNA from Whole Blood Using l0 Chelating Solid Phase Material One (1) pL of neat TRITON X-100 was added to one hundred (100) ~L of whole blood. The, solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (approximately 5 seconds every 20 seconds).
The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415 D centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) p,L
of concentrated material at the bottom of the centrifuge tube. This material was transferred to a ~M No. 2271 EMPORE Extraction Chelating Disk prepared as described in Example 1 G, making sure that the material was evenly distributed on the surface of the disk. The material was allowed to dry on the disk for about 2-S minutes until the intense red color became darker. Twelve (12) p,L of 0.083 M NaOH was added to the extraction disk. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 ~,L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 p,L aliquot was removed and added to 10 p.L of 40 mM TRIS-HCI (pH 7.4).
Example 16: Procedure for Isolation of Genomic DNA from Whole Blood Using Chelating Solid Phase Material 3o One (1) p.L of neat TRITON X-100 was added to one hundred (100) ~L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (approximately 5 seconds every 20 seconds).
The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415 D centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) p,L
of concentrated material at the bottom of the centrifuge tube. This material was transferred to a 3M No. 2271 EMPORE Extraction Chelating Disk prepared as described in Example 1H, making sure that the material was evenly distributed on the surface of the disk. The material was allowed to dry on the disk for about 2-5 minutes until the intense red color became darker. Twelve (12) p.L of 0.083 M NaOH was added to the extraction to disk. The solution was mixed up and down 2-3 times in a pipette tip and removed after mixing (although less than 12 p.L might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 p,L aliquot was removed and added to 10 wL of 40 mM TRIS-HCl (pH 7.4).
Example 17: Procedure for Isolation of Genomic DNA from Whole Blood Using Chelating Solid Phase Material One (1) p,L of neat TRITON X-100 was added to one hundred (100) p.L of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 2o minutes, vortexing the solution intermittently (approximately 5 seconds every 20 seconds).
The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415 D centrifuge at 400 rcf for about 10 minutes. The supernatant was separated and discarded, leaving about two (2) p,L
of concentrated material at the bottom of the centrifuge tube. This material was transferred to a 3M No. 2271 EMPORE Extraction Chelating Disk prepared as described in Example 1J, making sure that the material was evenly distributed on the surface of the disk. The material was allowed to dry on the disk for about 2-S minutes until the intense red color became darker. Twelve (12) p,L of 0.083 M NaOH was added to the extraction disk. The solution was mixed up and down 2-3 times in a pipette tip and removed after 3o mixing (although less than 12 pL might be obtained). The color of the sample removed from the membrane varied from colorless to faint green. If the solution was foamy, it was spun down at 4,000 revolutions per minute (rpm) for 1 minute. A 2 ~,L aliquot was removed and added to 10 pL of 40 mM TRIS-HCl (pH 7.4).
Example 18: Procedure for Isolation of Genomic DNA from Whole Blood Using Ammoniated Chelating Solid Phase Material with TRITON X-100 Mounted on a Microfluidic Device Two (2) ~.L of neat TRITON X-100 was added to one hundred (100) wL of whole blood. The solution was incubated at room temperature (approximately 21°C) for about 5 minutes, vortexing the solution intermittently (for approximately 5 seconds every 20 l0 seconds). The solution was investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415D
centrifuge at 400 rcf for about 2 min. The solution from the top was discarded leaving 4 ~,L at the bottom of the tube. To 4 p,L of concentrated material, 4 pL of RNase water was added, and the sample was mixed thoroughly. The sample then was placed into an injection port on a microfluidic device as described in Applicants' Assignee's copending U.S. Patent Application Serial No. 10/734,682, filed December 12, 2003 (Figure 1). The solution was spun at 500 rpm for 5 min to a clean up chamber containing a 3M
No. 2271 EMPORE Extraction Chelating Disk prepared as described in Example 1B using 10%
TRITON X-100 instead of 1% TRITON X-100 as a loading solution. A five (5) pL
2o aliquot of 0.1 M NaOH was added to a different injection port and was spun at 1000 rpm for 5 min to the clean up chamber described above. A ten (10) ~,L aliquot of 0.1 M NaOH
was added to the same injection port, and the solution was spun down at 2500 rpm for 10 min. A 5 p.L aliquot was removed and added to 1.5 ~,L of 1 M TRIS-HCl (pH
7.4).
Example 19: Procedure for Isolation of Genomic DNA from Whole Blood with the Use of a Chelating Solid Phase Material One hundred (100) pL of whole blood was added to one hundred (100) p.L 2%
TRITON X-100. The solution was mixed thoroughly, and then investigated to make sure that it was transparent before proceeding to the next step. The solution was spun in an Eppendorf Model 5415D centrifuge at 400 rcf for 2 min. A 95 p.L aliquot of the solution from the top was removed and discarded. Last five (5) p,L containing concentrated material was placed onto a 3M No. 2271 EMPORE Extraction Chelating Disk prepared as described in Example 1B using 10% TRITON X-100 instead of 1% TRITON X-100 as a loading solution. After the solution had soaked into the disk, the sample was extracted with a twenty (20) pL aliquot of 0.1 M NaOH. The solution was briefly spun in an Eppendorf Model 5415D centrifuge at 400 rc~ An aliquot of eleven (11) p.L of sample was heated for 3 min at 95°C, and then added to three (3) pL of 1 M
TRIS-HCl (pH 7.4).
Example 20: Procedure for Isolation of Genomic DNA from Whole Blood Five (5) p,L of whole blood was added to the ten (10) p.L of 10 mM NaOH. After l0 min incubation, the sample was heated for 3 min at 95°C. A 5 pL
aliquot of 16 mM TRIS-HCl (pH 7.4) was added to 10 p,L of sample.
RESULTS
Table 3 reports results that were obtained on ABI 7700 QPCR Machine (Applera, 15 Foster City, CA) following the instructions in QuantiTech SYBR Green PCR
Handbook on p.10-12 for preparation of a 10 p,L PCR sample (2 p,L of sample in 10 pL
SYBR Green Master Mix, 4 p,L ~i-actin, 4 p.L of water) for Examples 1-17, 19, and 20;
Results for Example 18 were obtained on LightCycler 2.0 (Roche Applied Science, Indianapolis, IN) following the instructions in LightCycler Factor V Leiden Mutation Kit's package insert 20 on p.2-3 for preparation of a 10 p,L PCR sample (2.5 p,L of sample in 5.5 p,L of RNase-free sterile water, 1 p,L of l Ox Factor V Leiden Reaction Mix and 1 p,L of lOx Factor V Leiden Mutation Detection Mix). One (1)% agarose gel (brightness of band- + faint, +++ bright) was run on Horizon 11-14 Electrophoresis Machine (Gibco BRL, Gaithersburg, MD).
Spectra measurements were run on a SpectraMax Plus384 spectrophotometer at 405 nm 25 (Molecular Devices Corporation, Sunnyvale, CA.). Two, three or four values for each sample represent duplicates, triplicates, or quadruplicates.
Table 3 Samples Ct Band 405 nm (avg) 1.5 ng/ pL human16.92 +++ -genomic DNA in 20.67 +++
0.1 M NaOH/40mM
TRIS-HCl buffer 1.5 ng/ ~,L human19.01 +++ 0 genomic DNA in 18.67 '~++
water 1.5 ng/ p,L human16.18 +++ -genomic DNA in 16.28 +++
water Example 4 17-20 +++ 0.24 (multiple samples'T+~-analyzed) Example 3 26, 28 + -Example 2 28, 27 none -Example SA 28 + 0.37 Example SB 17,18 +++ 0.24 +++
Example SC 24 ++ -26 ++
Example 6A 23 + 0 24 +
Example 6B 17 +++ 0 18 +++
Example 6C 21 ++ 0 21 ++
Examples 7A and - - 2.63 Mix I diluted 1:36 Examples 7A and - - 0.38 Mix I diluted 1:360 Examples 7A and - - 0.036 Mix I diluted 1:3600 Examples 7A and - - 0 Mix I diluted 1:36000 Example 8A 22 +++
Example 8B 22 ++
Example 8C 20 Example 9A 21 - 0.019 19 0.007 Example 9B 25 - 0.029 28 0.074 Example 10 26.86 +++
26.11 -Example 11 24.87 - -20.38 Example 12 26.74 - -26.04 Example 13 26.01 - 0.245 Example 14 19.82 - 0.377 Example 15 27.92 30.04 Example 16 - - 0.07 Example 17 - - 0.918 Example 18 27.39 (bench - -control 27.08) Example 19* 23.72, 24.03 - -Example 20* 30.35, 30.56 - -*Positive Control for Examples 19-20 was DNA extracted from two hundred (200) p,L of whole blood following "Blood and Body Fluid Spin Protocol" described in QIAamp DNA
Blood Mini Kit Handbook p. 27, eluting in 200 p,L of water and had Ct value of 23.
Negative Control (NTC or no template control) did not amplify in these experiments.
Various modifications and alterations to this invention will become apparent to those skilled in the art without departing from the scope of this invention.
It should be understood that this invention is not intended to be unduly limited by the illustrative l0 embodiments and examples set forth herein and that such examples and embodiments are presented by way of example only with the scope of the invention intended to be limited only by the claims set forth herein as follows.
Claims (47)
1. A method of isolating nucleic acid from a sample, the method comprising:
providing a sample comprising nucleic acid-containing material and inhibitors;
forming a concentrated region of the sample; wherein the concentrated region of the sample comprises nucleic acid-containing material and inhibitors;
substantially separating the concentrated region from a less concentrated region of the sample;
contacting the separated concentrated region of the sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
providing a sample comprising nucleic acid-containing material and inhibitors;
forming a concentrated region of the sample; wherein the concentrated region of the sample comprises nucleic acid-containing material and inhibitors;
substantially separating the concentrated region from a less concentrated region of the sample;
contacting the separated concentrated region of the sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
2. A method of isolating nucleic acid from a sample, the method comprising:
providing a sample comprising nucleic acid-containing material and cells containing inhibitors;
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample comprising nucleic acid-containing material and inhibitors;
forming a concentrated region of the lysed sample; wherein the concentrated region of the lysed sample comprises nucleic acid-containing material and inhibitors;
substantially separating the concentrated region from a less concentrated region of the lysed sample;
contacting the separated concentrated region of the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
providing a sample comprising nucleic acid-containing material and cells containing inhibitors;
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample comprising nucleic acid-containing material and inhibitors;
forming a concentrated region of the lysed sample; wherein the concentrated region of the lysed sample comprises nucleic acid-containing material and inhibitors;
substantially separating the concentrated region from a less concentrated region of the lysed sample;
contacting the separated concentrated region of the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
3. A method of isolating nucleic acid from a sample, the method comprising:
providing a sample comprising cells containing inhibitors and cells containing nuclei;
contacting the biological sample with a nonionic surfactant under conditions effective to break cell membranes and release nuclei and inhibitors and form a lysed sample;
forming a concentrated region of the lysed sample; wherein the concentrated region of the sample comprises nuclei and inhibitors;
substantially separating the concentrated region of the lysed sample from the less concentrated region of the sample;
contacting the separated concentrated region of the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally further lysing the nuclei to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nuclei and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
providing a sample comprising cells containing inhibitors and cells containing nuclei;
contacting the biological sample with a nonionic surfactant under conditions effective to break cell membranes and release nuclei and inhibitors and form a lysed sample;
forming a concentrated region of the lysed sample; wherein the concentrated region of the sample comprises nuclei and inhibitors;
substantially separating the concentrated region of the lysed sample from the less concentrated region of the sample;
contacting the separated concentrated region of the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally further lysing the nuclei to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nuclei and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
4. A method of isolating nucleic acid from a sample, the method comprising:
providing a microfluidic device comprising a loading chamber, a valued process chamber, and a separation chamber comprising a solid phase material;
providing a sample comprising nucleic acid-containing material and inhibitors;
placing the sample in the loading chamber;
transferring the sample to a valued process chamber;
forming a concentrated region of the sample in the valued process chamber, wherein the concentrated region of the sample comprises nucleic acid-containing material and inhibitors;
substantially separating the concentrated region from a less concentrated region of the sample;
transferring the separated concentrated region of the sample to the separation chamber for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
providing a microfluidic device comprising a loading chamber, a valued process chamber, and a separation chamber comprising a solid phase material;
providing a sample comprising nucleic acid-containing material and inhibitors;
placing the sample in the loading chamber;
transferring the sample to a valued process chamber;
forming a concentrated region of the sample in the valued process chamber, wherein the concentrated region of the sample comprises nucleic acid-containing material and inhibitors;
substantially separating the concentrated region from a less concentrated region of the sample;
transferring the separated concentrated region of the sample to the separation chamber for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
5. A method of isolating nucleic acid from a sample, the method comprising:
providing a microfluidic device comprising a loading chamber, a valved process chamber, and a separation chamber comprising a solid phase material;
providing a sample comprising nucleic acid-containing material and cells containing inhibitors;
placing the sample in the loading chamber;
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample comprising nucleic acid-containing material and inhibitors;
transferring the lysed sample to a valued process chamber;
forming a concentrated region of the lysed sample in the valued process chamber, wherein the concentrated region of the lysed sample comprises nucleic acid-containing material and inhibitors;
substantially separating the concentrated region from a less concentrated region of the lysed sample;
transferring the separated concentrated region of the lysed sample to the separation chamber for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
providing a microfluidic device comprising a loading chamber, a valved process chamber, and a separation chamber comprising a solid phase material;
providing a sample comprising nucleic acid-containing material and cells containing inhibitors;
placing the sample in the loading chamber;
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample comprising nucleic acid-containing material and inhibitors;
transferring the lysed sample to a valued process chamber;
forming a concentrated region of the lysed sample in the valued process chamber, wherein the concentrated region of the lysed sample comprises nucleic acid-containing material and inhibitors;
substantially separating the concentrated region from a less concentrated region of the lysed sample;
transferring the separated concentrated region of the lysed sample to the separation chamber for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
6. The method of claim 5 wherein the nucleic acid-containing material comprises nuclei and the method comprises separating at least a portion of the nuclei from the solid phase material.
7. The method of claim 5 wherein separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material comprises transferring the nucleic acid-containing material and/or nucleic acid to an amplification reaction chamber.
8. The method of claim 7 further comprising subjecting the nuclei and/or nucleic acid to an amplification reaction.
9. The method of claim 5 wherein separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material comprises eluting at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material with an eluting reagent, optionally heating the nucleic acid-containing material and/or nuclei, and optionally adjusting the pH.
10. A method of isolating nucleic acid from a sample, the method comprising:
providing a microfluidic device comprising a loading chamber, a valued process chamber, and a separation chamber comprising a solid phase material;
nuclei;
providing a sample comprising cells containing inhibitors and cells containing placing the sample in the loading chamber;
contacting the sample with a nonionic surfactant under conditions effective to break cell membranes and release nuclei and inhibitors to form a lysed sample;
transferring the lysed sample to a valued process chamber;
forming a concentrated region of the lysed sample in the valued process chamber, wherein the concentrated region of the lysed sample comprises nuclei and inhibitors;
substantially separating the concentrated region from a less concentrated region of the lysed sample;
transferring the separated concentrated region of the lysed sample to the separation chamber for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally further lysing the nuclei to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nuclei and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
providing a microfluidic device comprising a loading chamber, a valued process chamber, and a separation chamber comprising a solid phase material;
nuclei;
providing a sample comprising cells containing inhibitors and cells containing placing the sample in the loading chamber;
contacting the sample with a nonionic surfactant under conditions effective to break cell membranes and release nuclei and inhibitors to form a lysed sample;
transferring the lysed sample to a valued process chamber;
forming a concentrated region of the lysed sample in the valued process chamber, wherein the concentrated region of the lysed sample comprises nuclei and inhibitors;
substantially separating the concentrated region from a less concentrated region of the lysed sample;
transferring the separated concentrated region of the lysed sample to the separation chamber for contact with the solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally further lysing the nuclei to release nucleic acid before, simultaneous with, or after contacting the separated concentrated region of the sample with the solid phase material; and separating at least a portion of the nuclei and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto.
11. The method of claim 10 wherein separating at least a portion of the nuclei and/or nucleic acid from the solid phase material comprises transferring the nuclei and/or nucleic acid to an amplification reaction chamber.
12. The method of claim 11 further comprising optionally heating the nuclei and/or nucleic acid, optionally adjusting the pH, and subjecting the nuclei and/or nucleic acid to an amplification reaction.
13. The method of claim 10 wherein placing the sample in the loading chamber occurs prior to contacting the sample with a nonionic surfactant.
14. The method of claim 10 wherein the loading chamber comprises pre-deposited nonionic surfactant and contacting the sample with a nonionic surfactant occurs upon placing the sample in the loading chamber.
15. The method of claim 10 wherein placing the sample in the loading chamber occurs after contacting the sample with a nonionic surfactant.
16. The method of claim 10 wherein contacting the sample with a nonionic surfactant occurs in a mixing chamber with sufficient mixing to break cell membranes and release nuclei and inhibitors to form a lysed sample.
17. The method of claim 10 wherein forming a concentrated region of the sample in the valued process chamber comprises centrifuging the sample in the valued process chamber.
18. The method of claim 17 wherein separating the concentrated region from a less concentrated region of the sample comprises transferring the less concentrated region of the sample through the valve to a waste bin.
19. The method of claim 10 wherein the coating reagent comprises a surfactant.
20. The method of claim 10 wherein separating at least a portion of the nuclei and/or nucleic acid from the solid phase material comprises eluting at least a portion of the nuclei and/or nucleic acid from the solid phase material with an eluting reagent.
21. The method of claim 20 wherein the eluting reagent is a lysing reagent.
22. The method of claim 10 wherein the further lysing step is carried out with a second lysing reagent.
23. A method of isolating nucleic acid from a sample, the method comprising:
providing a sample comprising nucleic acid and inhibitors;
contacting the sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally lysing nucleic acid-containing material, if present, to release nucleic acid before, simultaneous with, or after contacting the lysed sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic surfactant.
providing a sample comprising nucleic acid and inhibitors;
contacting the sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally lysing nucleic acid-containing material, if present, to release nucleic acid before, simultaneous with, or after contacting the lysed sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic surfactant.
24. A method of isolating nucleic acid from a sample, the method comprising:
providing a sample comprising nucleic acid-containing material and cells containing inhibitors;
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample comprising nucleic acid-containing material and inhibitors;
contacting the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the lysed sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic surfactant.
providing a sample comprising nucleic acid-containing material and cells containing inhibitors;
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample comprising nucleic acid-containing material and inhibitors;
contacting the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the lysed sample with the solid phase material; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic surfactant.
25. A method of isolating nucleic acid from a sample, the method comprising:
providing a sample comprising nucleic acid-containing material and cells containing inhibitors;
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample comprising nucleic acid-containing material and inhibitors;
contacting the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
separating at least a portion of the nucleic acid-containing material from the solid phase material having at least a portion of the inhibitors adhered thereto;
and after separating the nucleic acid-containing material, further lysing the nucleic acid-containing material to release nucleic acid.
providing a sample comprising nucleic acid-containing material and cells containing inhibitors;
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample comprising nucleic acid-containing material and inhibitors;
contacting the lysed sample with a solid phase material to preferentially adhere at least a portion of the inhibitors to the solid phase material, wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof;
separating at least a portion of the nucleic acid-containing material from the solid phase material having at least a portion of the inhibitors adhered thereto;
and after separating the nucleic acid-containing material, further lysing the nucleic acid-containing material to release nucleic acid.
26. The method of claim 25 wherein the nucleic acid-containing material comprises nuclei.
27. The method of claim 25 wherein the coating reagent comprises a surfactant.
28. A method of isolating nucleic acid from a sample, the method comprising:
providing a microfluidic device comprising a loading chamber and a separation chamber comprising a solid phase material;
providing a sample comprising nucleic acid-containing material and cells containing inhibitors;
placing the sample in the loading chamber;
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample comprising nucleic acid-containing material and inhibitors;
transferring the lysed sample to the separation chamber to contact the solid phase material and to preferentially adhere at least a portion of the inhibitors to the solid phase material;
optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the sample with the solid phase material;
wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic surfactant.
providing a microfluidic device comprising a loading chamber and a separation chamber comprising a solid phase material;
providing a sample comprising nucleic acid-containing material and cells containing inhibitors;
placing the sample in the loading chamber;
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample comprising nucleic acid-containing material and inhibitors;
transferring the lysed sample to the separation chamber to contact the solid phase material and to preferentially adhere at least a portion of the inhibitors to the solid phase material;
optionally further lysing the nucleic acid-containing material to release nucleic acid before, simultaneous with, or after contacting the sample with the solid phase material;
wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof; and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material having at least a portion of the inhibitors adhered thereto by eluting with an eluting reagent, with the proviso that the eluting reagent is not a nonionic surfactant.
29. A method of isolating nucleic acid from a sample, the method comprising:
providing a microfluidic device comprising a loading chamber and a separation chamber comprising a solid phase material;
providing a sample comprising nucleic acid-containing material and cells containing inhibitors;
placing the sample in the loading chamber;
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample comprising nucleic acid-containing material and inhibitors;
transferring the lysed sample to the separation chamber to contact the solid phase material and to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof; and separating at least a portion of the nucleic acid-containing material from the solid phase material having at least a portion of the inhibitors adhered thereto;
and after separating the nucleic acid-containing material, further lysing the nucleic acid-containing material to release nucleic acid.
providing a microfluidic device comprising a loading chamber and a separation chamber comprising a solid phase material;
providing a sample comprising nucleic acid-containing material and cells containing inhibitors;
placing the sample in the loading chamber;
contacting the sample with a first lysing reagent under conditions effective to break cell membranes and release inhibitors and form a lysed sample comprising nucleic acid-containing material and inhibitors;
transferring the lysed sample to the separation chamber to contact the solid phase material and to preferentially adhere at least a portion of the inhibitors to the solid phase material; wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof; and separating at least a portion of the nucleic acid-containing material from the solid phase material having at least a portion of the inhibitors adhered thereto;
and after separating the nucleic acid-containing material, further lysing the nucleic acid-containing material to release nucleic acid.
30. The method of claim 29 wherein the nucleic acid-containing material comprises nuclei.
31. The method of claim 29 wherein separating the nucleic acid-containing material and/or nucleic acid from the solid phase material comprises transferring the nucleic acid-containing material and/or nucleic acid to an amplification reaction chamber.
32. The method of claim 31 further comprising subjecting the nucleic acid-containing material and/or nucleic acid to an amplification reaction.
33. The method of claim 29 wherein placing the sample in the loading chamber occurs prior to contacting the sample with a first lysing reagent.
34. The method of claim 29 wherein the loading chamber comprises the first lysing reagent and contacting the sample with a first lysing reagent occurs upon placing the sample in the loading chamber.
35. The method of claim 29 wherein placing the sample in the loading chamber occurs after contacting the sample with a first lysing reagent.
36. The method of claim 29 wherein forming a concentrated region of the sample in the valued process chamber comprises centrifuging the sample in the process chamber.
37. The method of claim 29 wherein the solid phase material comprises a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material, wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte a selectively permeable polymeric barrier, and combinations thereof.
38. The method of claim 29 wherein the first lysing reagent is a nonionic surfactant.
39. The method of claim 29 wherein separating comprises eluting at least a portion of the nucleic acid-containing material from the solid phase material with an eluting reagent.
40. A kit for isolating nucleic acid from a sample, the kit comprising:
a first lysing reagent;
a solid phase material comprising a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric burner, and combinations thereof; and instructions for lysing a sample and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material according to the method of claim 2.
a first lysing reagent;
a solid phase material comprising a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric burner, and combinations thereof; and instructions for lysing a sample and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material according to the method of claim 2.
41. A kit for isolating nucleic acid from a sample, the kit comprising:
a first lysing reagent;
a solid phase material comprising a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof; and instructions for lysing a sample and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material according to the method of claim 24.
a first lysing reagent;
a solid phase material comprising a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier, and combinations thereof; and instructions for lysing a sample and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material according to the method of claim 24.
42. A kit for isolating nucleic acid from a sample, the kit comprising:
a nonionic surfactant;
a solid phase material comprising a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier and combinations thereof; and instructions for lysing a sample and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material according to the method of claim 3.
a nonionic surfactant;
a solid phase material comprising a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier and combinations thereof; and instructions for lysing a sample and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material according to the method of claim 3.
43. A kit for isolating nucleic acid from a sample, the kit comprising:
a nonionic surfactant;
a solid phase material comprising a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier and combinations thereof; and instructions for lysing a sample and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material according to the method of claim 25.
a nonionic surfactant;
a solid phase material comprising a polytetrafluoroethylene fibril matrix, sorptive particles enmeshed in the matrix, and a coating reagent coated on the solid phase material;
wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier and combinations thereof; and instructions for lysing a sample and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material according to the method of claim 25.
44. A kit for isolating nucleic acid from a sample, the kit comprising:
a first lysing reagent;
a microfluidic device comprising a loading chamber, a valued process chamber, and a separation chamber comprising a solid phase material; wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier and combinations thereof; and instructions for lysing a sample and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material according to the method of claim 5.
a first lysing reagent;
a microfluidic device comprising a loading chamber, a valued process chamber, and a separation chamber comprising a solid phase material; wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier and combinations thereof; and instructions for lysing a sample and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material according to the method of claim 5.
45. A kit for isolating nucleic acid from a sample, the kit comprising:
a first lysing reagent;
a microfluidic device comprising a loading chamber, a valued process chamber, and a separation chamber comprising a solid phase material; wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier and combinations thereof; and instructions for lysing a sample and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material according to the method of claim 28.
a first lysing reagent;
a microfluidic device comprising a loading chamber, a valued process chamber, and a separation chamber comprising a solid phase material; wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier and combinations thereof; and instructions for lysing a sample and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material according to the method of claim 28.
46. A kit for isolating nucleic acid from a sample, the kit comprising:
a nonionic surfactant;
a microfluidic device comprising a loading chamber, a valued process chamber, and a separation chamber comprising a solid phase material; wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier and combinations thereof; and instructions for lysing a sample and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material according to the method of claim 10.
a nonionic surfactant;
a microfluidic device comprising a loading chamber, a valued process chamber, and a separation chamber comprising a solid phase material; wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier and combinations thereof; and instructions for lysing a sample and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material according to the method of claim 10.
47. A kit for isolating nucleic acid from a sample, the kit comprising:
a nonionic surfactant;
a microfluidic device comprising a loading chamber, a valued process chamber, and a separation chamber comprising a solid phase material; wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier and combinations thereof; and instructions for lysing a sample and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material according to the method of claim 29.
a nonionic surfactant;
a microfluidic device comprising a loading chamber, a valued process chamber, and a separation chamber comprising a solid phase material; wherein the solid phase material comprises capture sites, a coating reagent coated on the solid phase material, or both; wherein the coating reagent is selected from the group consisting of a surfactant, a strong base, a polyelectrolyte, a selectively permeable polymeric barrier and combinations thereof; and instructions for lysing a sample and separating at least a portion of the nucleic acid-containing material and/or nucleic acid from the solid phase material according to the method of claim 29.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US53252303P | 2003-12-24 | 2003-12-24 | |
US60/532,523 | 2003-12-24 | ||
US10/852,645 | 2004-05-24 | ||
US10/852,645 US20050142571A1 (en) | 2003-12-24 | 2004-05-24 | Methods for nucleic acid isolation and kits using solid phase material |
PCT/US2004/035367 WO2005068628A2 (en) | 2003-12-24 | 2004-10-25 | Methods for nucleic acid isolation and kits using solid phase material |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2551462A1 true CA2551462A1 (en) | 2005-07-28 |
Family
ID=34704322
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002551462A Abandoned CA2551462A1 (en) | 2003-12-24 | 2004-10-25 | Methods for nucleic acid isolation and kits using solid phase material |
Country Status (6)
Country | Link |
---|---|
US (1) | US20050142571A1 (en) |
EP (1) | EP1697514A2 (en) |
JP (1) | JP2007516724A (en) |
AU (1) | AU2004313914A1 (en) |
CA (1) | CA2551462A1 (en) |
WO (1) | WO2005068628A2 (en) |
Families Citing this family (43)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7192560B2 (en) * | 2001-12-20 | 2007-03-20 | 3M Innovative Properties Company | Methods and devices for removal of organic molecules from biological mixtures using anion exchange |
US6889468B2 (en) | 2001-12-28 | 2005-05-10 | 3M Innovative Properties Company | Modular systems and methods for using sample processing devices |
US7322254B2 (en) * | 2003-12-12 | 2008-01-29 | 3M Innovative Properties Company | Variable valve apparatus and methods |
US20050130177A1 (en) * | 2003-12-12 | 2005-06-16 | 3M Innovative Properties Company | Variable valve apparatus and methods |
US20050142570A1 (en) * | 2003-12-24 | 2005-06-30 | 3M Innovative Properties Company | Methods for nucleic acid isolation and kits using a microfluidic device and sedimenting reagent |
US7939249B2 (en) | 2003-12-24 | 2011-05-10 | 3M Innovative Properties Company | Methods for nucleic acid isolation and kits using a microfluidic device and concentration step |
US7387877B2 (en) * | 2004-04-06 | 2008-06-17 | Oro Grande Technology | Bio-sensor and bio-sensor reporting system |
KR100647315B1 (en) * | 2005-02-02 | 2006-11-23 | 삼성전자주식회사 | Method for amplifying nucleic acids using a silanized solid support |
US7323660B2 (en) | 2005-07-05 | 2008-01-29 | 3M Innovative Properties Company | Modular sample processing apparatus kits and modules |
US7754474B2 (en) | 2005-07-05 | 2010-07-13 | 3M Innovative Properties Company | Sample processing device compression systems and methods |
US7763210B2 (en) | 2005-07-05 | 2010-07-27 | 3M Innovative Properties Company | Compliant microfluidic sample processing disks |
US20080280290A1 (en) * | 2005-08-19 | 2008-11-13 | Dawson Elliott P | Method and Device for the Collection and Isolation of Nucleic Acid |
US20080121591A1 (en) * | 2006-11-29 | 2008-05-29 | Canon U.S. Life Sciences, Inc. | Device for nucleic acid preparation |
ES2453379T3 (en) * | 2007-04-25 | 2014-04-07 | 3M Innovative Properties Company | Supported reagents, methods and devices |
US20100062421A1 (en) * | 2007-04-25 | 2010-03-11 | Wensheng Xia | Compositions, methods, and devices for isolating biological materials |
US8304185B2 (en) | 2009-07-17 | 2012-11-06 | Canon U.S. Life Sciences, Inc. | Methods and systems for DNA isolation on a microfluidic device |
JP2012513768A (en) | 2008-12-31 | 2012-06-21 | スリーエム イノベイティブ プロパティズ カンパニー | Sampling apparatus and method for concentrating microorganisms |
EP2379226B1 (en) * | 2008-12-31 | 2016-04-20 | 3M Innovative Properties Company | Live bioload detection using microparticles |
JP5721704B2 (en) * | 2009-06-12 | 2015-05-20 | マイクロニクス, インコーポレイテッド | Rehydratable matrix for dry storage of TAQ polymerase in microfluidic devices |
CN102803507B (en) | 2009-06-12 | 2016-05-25 | 精密公司 | The composition and the method that store in the dehydration of plate reactant for microfluidic device |
JP5815518B2 (en) | 2009-07-17 | 2015-11-17 | キヤノン ユー.エス. ライフ サイエンシズ, インコーポレイテッドCanon U.S. Life Sciences, Inc. | Method and system for isolating DNA in a microfluidic device |
USD638951S1 (en) | 2009-11-13 | 2011-05-31 | 3M Innovative Properties Company | Sample processing disk cover |
US8834792B2 (en) | 2009-11-13 | 2014-09-16 | 3M Innovative Properties Company | Systems for processing sample processing devices |
USD638550S1 (en) | 2009-11-13 | 2011-05-24 | 3M Innovative Properties Company | Sample processing disk cover |
USD667561S1 (en) | 2009-11-13 | 2012-09-18 | 3M Innovative Properties Company | Sample processing disk cover |
US9284593B2 (en) | 2009-12-30 | 2016-03-15 | 3M Innovative Properties Company | Live bioload detection using microparticles |
JP2014507669A (en) | 2011-03-08 | 2014-03-27 | ユニベルシテ・ラバル | Fluid centripetal device |
EP2709762B1 (en) | 2011-05-18 | 2021-03-31 | DiaSorin S.p.A. | Systems and methods for detecting the presence of a selected volume of material in a sample processing device |
MX336651B (en) | 2011-05-18 | 2016-01-27 | 3M Innovative Properties Co | Systems and methods for valving on a sample processing device. |
USD672467S1 (en) | 2011-05-18 | 2012-12-11 | 3M Innovative Properties Company | Rotatable sample processing disk |
CN103547370A (en) | 2011-05-18 | 2014-01-29 | 3M创新有限公司 | Systems and methods for volumetric metering on a sample processing device |
CN102796727B (en) * | 2011-05-24 | 2014-06-18 | 博奥生物有限公司 | Method for extracting nucleic acid of gram positive bacteria |
US9610579B2 (en) | 2014-01-07 | 2017-04-04 | Daktari Diagnostics, Inc. | Fluid delivery devices, systems, and methods |
US20160123975A1 (en) * | 2014-11-03 | 2016-05-05 | Daktari Diagnostics, Inc. | Mesh Microfluidic Mixing Chamber |
WO2016109503A2 (en) * | 2014-12-31 | 2016-07-07 | Peaslee Graham F | Method for detecting fluorinated chemicals in liquid |
US11207681B2 (en) | 2015-07-17 | 2021-12-28 | Delta Electronics, Inc. | Method for extracting nucleic acid and extraction cassette thereof |
TWI591182B (en) | 2015-07-17 | 2017-07-11 | 台達電子工業股份有限公司 | Nucleic acid extracting device |
US11491482B2 (en) | 2015-07-17 | 2022-11-08 | Delta Electronics, Inc. | Method for extracting nucleic acid and extraction cassette thereof |
USD799715S1 (en) | 2015-10-23 | 2017-10-10 | Gene POC, Inc. | Fluidic centripetal device |
GB201703383D0 (en) | 2017-03-02 | 2017-04-19 | Gargle Tech Ltd | Testing for particulates |
CA3086996A1 (en) | 2017-12-27 | 2019-07-04 | Toray Industries, Inc. | Method for collecting nucleic acid |
US11680877B2 (en) | 2018-09-05 | 2023-06-20 | Hero Scientific Ltd. | Testing for particulates |
CA3202405A1 (en) | 2021-01-06 | 2022-07-14 | Zvi Feldman | Filtration sampling devices |
Family Cites Families (44)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3157635A (en) * | 1960-08-05 | 1964-11-17 | Takeda Chemical Industries Ltd | Sulfonic acid cation exchange resin purification of nucleotides |
US4399009A (en) * | 1981-01-19 | 1983-08-16 | Oronzio Denora Impianti Elettrochimici S.P.A. | Electrolytic cell and method |
US4839296A (en) * | 1985-10-18 | 1989-06-13 | Chem-Elec, Inc. | Blood plasma test method |
US5079155A (en) * | 1987-03-02 | 1992-01-07 | E. I. Du Pont De Nemours And Company | Fluorocarbon polymer support for chromatographic separations, diagnostic assays and enzyme immobilization |
US5284940A (en) * | 1990-11-14 | 1994-02-08 | Hri Research, Inc. | Preparation for nucleic acid samples |
CA2059398C (en) * | 1991-02-07 | 1999-05-25 | Craig G. Markell | Solid phase extraction medium |
US6093558A (en) * | 1991-07-25 | 2000-07-25 | Edge Biosystems, Inc. | Binding protein of biologically active compositions to an adhesive formulation on a substrate |
DE69316231T2 (en) * | 1992-10-21 | 1998-08-20 | Cornell Res Foundation Inc | SELECTIVE, CHEMICAL MODIFICATION OF POROUS MATERIALS |
US6780818B2 (en) * | 1994-02-02 | 2004-08-24 | The Regents Of The University Of California | Quantitative organic vapor-particle sampler |
SE9500183D0 (en) * | 1995-01-20 | 1995-01-20 | Pharmacia Biotech Ab | Method for the purification of short nucleic acids |
US5705059A (en) * | 1995-02-27 | 1998-01-06 | Miltenyi; Stefan | Magnetic separation apparatus |
US6168948B1 (en) * | 1995-06-29 | 2001-01-02 | Affymetrix, Inc. | Miniaturized genetic analysis systems and methods |
US5856174A (en) * | 1995-06-29 | 1999-01-05 | Affymetrix, Inc. | Integrated nucleic acid diagnostic device |
DE19530132C2 (en) * | 1995-08-16 | 1998-07-16 | Max Planck Gesellschaft | Process for the purification, stabilization or isolation of nucleic acids from biological materials |
US5620869A (en) * | 1995-09-28 | 1997-04-15 | Becton, Dickinson And Company | Methods for reducing inhibition of nucleic acid amplification reactions |
JP3818689B2 (en) * | 1996-01-16 | 2006-09-06 | 富士写真フイルム株式会社 | Aqueous dispersion of core / shell composite particles having colloidal silica as the core and organic polymer as the shell, and method for producing the same |
US5837203A (en) * | 1996-04-09 | 1998-11-17 | Sievers Instruments, Inc. | Device to alternately supply a fluid to an analyzer |
US6074827A (en) * | 1996-07-30 | 2000-06-13 | Aclara Biosciences, Inc. | Microfluidic method for nucleic acid purification and processing |
US6143248A (en) * | 1996-08-12 | 2000-11-07 | Gamera Bioscience Corp. | Capillary microvalve |
WO1998028623A1 (en) * | 1996-12-20 | 1998-07-02 | Gamera Bioscience Corporation | An affinity binding-based system for detecting particulates in a fluid |
AU7591998A (en) * | 1997-05-23 | 1998-12-11 | Gamera Bioscience Corporation | Devices and methods for using centripetal acceleration to drive fluid movement in a microfluidics system |
US6241980B1 (en) * | 1997-11-04 | 2001-06-05 | Becton, Dickinson And Company | Sample processing method using ion exchange resin |
EP1042061A1 (en) * | 1997-12-24 | 2000-10-11 | Cepheid | Integrated fluid manipulation cartridge |
EP1092144A1 (en) * | 1998-06-29 | 2001-04-18 | Evotec BioSystems AG | Method and device for manipulating particles in microsystems |
WO2000019834A1 (en) * | 1998-10-08 | 2000-04-13 | Fuji Oil Co., Ltd. | Chocolate compositions and utilization thereof |
WO2000079285A2 (en) * | 1999-06-18 | 2000-12-28 | Gamera Bioscience Corporation | Devices and methods for the performance of miniaturized homogeneous assays |
US6383783B1 (en) * | 1999-09-21 | 2002-05-07 | 3M Innovative Properties Company | Nucleic acid isolation by adhering to hydrophobic solid phase and removing with nonionic surfactant |
DE60042561D1 (en) * | 1999-10-27 | 2009-08-27 | 3M Innovative Properties Co | FLUOROCHEMICAL SULPHONAMIDE TENSIDES |
WO2001062887A1 (en) * | 2000-02-23 | 2001-08-30 | Zyomyx, Inc. | Chips having elevated sample surfaces |
WO2001087486A2 (en) * | 2000-05-15 | 2001-11-22 | Tecan Trading Ag | Microfluidics devices and methods for performing cell based assays |
US6734401B2 (en) * | 2000-06-28 | 2004-05-11 | 3M Innovative Properties Company | Enhanced sample processing devices, systems and methods |
US6627159B1 (en) * | 2000-06-28 | 2003-09-30 | 3M Innovative Properties Company | Centrifugal filling of sample processing devices |
US6504021B2 (en) * | 2000-07-05 | 2003-01-07 | Edge Biosystems, Inc. | Ion exchange method for DNA purification |
US6537502B1 (en) * | 2000-07-25 | 2003-03-25 | Harvard Apparatus, Inc. | Surface coated housing for sample preparation |
US6919058B2 (en) * | 2001-08-28 | 2005-07-19 | Gyros Ab | Retaining microfluidic microcavity and other microfluidic structures |
ATE477054T1 (en) * | 2001-09-17 | 2010-08-15 | Gyros Patent Ab | FUNCTIONAL UNIT ALLOWING CONTROLLED FLOW IN A MICROFLUID DEVICE |
US7192560B2 (en) * | 2001-12-20 | 2007-03-20 | 3M Innovative Properties Company | Methods and devices for removal of organic molecules from biological mixtures using anion exchange |
US7347976B2 (en) * | 2001-12-20 | 2008-03-25 | 3M Innovative Properties Company | Methods and devices for removal of organic molecules from biological mixtures using a hydrophilic solid support in a hydrophobic matrix |
US6532997B1 (en) * | 2001-12-28 | 2003-03-18 | 3M Innovative Properties Company | Sample processing device with integral electrophoresis channels |
US7981600B2 (en) * | 2003-04-17 | 2011-07-19 | 3M Innovative Properties Company | Methods and devices for removal of organic molecules from biological mixtures using an anion exchange material that includes a polyoxyalkylene |
US20050130177A1 (en) * | 2003-12-12 | 2005-06-16 | 3M Innovative Properties Company | Variable valve apparatus and methods |
US7939249B2 (en) * | 2003-12-24 | 2011-05-10 | 3M Innovative Properties Company | Methods for nucleic acid isolation and kits using a microfluidic device and concentration step |
US20050142570A1 (en) * | 2003-12-24 | 2005-06-30 | 3M Innovative Properties Company | Methods for nucleic acid isolation and kits using a microfluidic device and sedimenting reagent |
US7727710B2 (en) * | 2003-12-24 | 2010-06-01 | 3M Innovative Properties Company | Materials, methods, and kits for reducing nonspecific binding of molecules to a surface |
-
2004
- 2004-05-24 US US10/852,645 patent/US20050142571A1/en not_active Abandoned
- 2004-10-25 WO PCT/US2004/035367 patent/WO2005068628A2/en active Application Filing
- 2004-10-25 EP EP04796359A patent/EP1697514A2/en not_active Withdrawn
- 2004-10-25 CA CA002551462A patent/CA2551462A1/en not_active Abandoned
- 2004-10-25 JP JP2006546980A patent/JP2007516724A/en not_active Withdrawn
- 2004-10-25 AU AU2004313914A patent/AU2004313914A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
WO2005068628A2 (en) | 2005-07-28 |
JP2007516724A (en) | 2007-06-28 |
US20050142571A1 (en) | 2005-06-30 |
AU2004313914A1 (en) | 2005-07-28 |
EP1697514A2 (en) | 2006-09-06 |
WO2005068628A3 (en) | 2005-09-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7939249B2 (en) | Methods for nucleic acid isolation and kits using a microfluidic device and concentration step | |
US20050142571A1 (en) | Methods for nucleic acid isolation and kits using solid phase material | |
US20050142570A1 (en) | Methods for nucleic acid isolation and kits using a microfluidic device and sedimenting reagent | |
EP1692283B1 (en) | Variable valve apparatus and methods | |
CA2501056C (en) | Methods and materials for using chemical compounds as a tool for nucleic acid storage on media of nucleic acid purification systems | |
US9464316B2 (en) | Method for isolating nucleic acids comprising the use of ethylene glycol multimers | |
CA2289943C (en) | Solid-phase nucleic acid isolation | |
AU2002318631B2 (en) | Method of purifying nucleic acid using nonwoven fabric and detection method | |
EP1216255B1 (en) | Method for nucleic acid isolation and kit | |
CN1918291A (en) | Methods for nucleic acid isolation and kits using a microfluidic device and sedimenting reagent |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FZDE | Discontinued |